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Abstract:

The present invention relates generally to stable formulations comprising
CTLA4Ig molecules, including lyophilized, and liquid formulations for
administration via various routes including, for example, routes such as
intravenous (IV) and subcutaneous (SC) for treating immune system
diseases and tolerance induction.

Claims:

1. A method for treating immune system diseases comprising administering
to a subject in need thereof an effective amount of a stable formulation
suitable for subcutaneous administration comprising at least 100 mg/ml
CTLA4Ig molecules, a sugar capable of stabilizing said formulation at a
concentration effective therefore and a pharmaceutically acceptable
aqueous carrier.

2. The method of claim 1 wherein immune system diseases are selected from
the group containing autoimmune diseases, immunoproliferative diseases,
and graft-related disorders.

7. The method of claim 6 wherein a rheumatic disease is rheumatoid
arthritis.

8. The method of claim 1 wherein the CTLA4Ig formulation further
comprises a pH range of from 6 to 8.

9. The method of claim 1 wherein the CTLA4Ig formulation further
comprises a viscosity of from 9 to 20 cps.

10. The method of claim 1 wherein the sugar suitable for stabilizing the
formulation is selected from sucrose, lactose, maltose, mannitol and
trehalose.

11. The method of claim 10 wherein the formulation is stabilized by a
sugar:protein ratio of 1.1:1 or higher.

12. The method of claim 10 wherein the sugar is sucrose.

13. The method of claim 1 wherein the CTLA4Ig formulation further
comprises a pharmaceutically acceptable buffer.

14. The method of claim 1 wherein the CTLA4Ig molecule has the amino acid
sequence shown in ID NO:2 starting at methionine at position 27 or
alanine at position 26 and ending at lysine at position 383 or glycine at
position 382.

15. A method for treating rheumatoid arthritis comprising administering
to a subject in need thereof an effective amount of a stable formulation
suitable for subcutaneous administration comprising the CTLA4Ig molecule
having the amino acid sequence shown in SEQ ID NO:2 starting at
methionine at position 27 or alanine at position 26 and ending at lysine
at position 383 or glycine at position 382 in an amount of about 125
mg/ml, sucrose in an amount of about 170 mg/ml, at least one buffering
agent, sterile water for injection and optionally a surfactant.

Description:

[0001] The present patent application is a divisional application of U.S.
Ser. No. 12/086,876 filed Dec. 19, 2006, now allowed, which claims the
priority of provisional application U.S. Ser. No. 60/752,150, filed on
Dec. 20, 2005, the contents of which are hereby incorporated by reference
in their entirety into this application

[0002] All patents, patent applications and publications cited herein are
hereby incorporated by reference in their entirety. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the state
of the art as known to those skilled therein as of the date of the
invention described and claimed herein.

FIELD OF THE INVENTION

[0003] The present invention relates generally to stable formulations
comprising CTLA4Ig molecules, including lyophilized, and liquid
formulations for administration via various routes including, for
example, routes such as intravenous (IV) and subcutaneous (SC).

BACKGROUND OF THE INVENTION

[0004] Over the past two decades, recombinant DNA technology has led to
the commercialization of many protein therapeutics. The most conventional
route of delivery for protein drugs has been intravenous (IV)
administration because of poor bioavailability by most other routes,
greater control during clinical administration, and faster pharmaceutical
development. For products that require frequent and chronic
administration, the alternate subcutaneous (SC) route of delivery is more
appealing. When coupled with pre-filled syringe and autoinjector device
technology, SC delivery allows for home administration and improved
compliance of administration.

[0005] Treatments with high doses of more than 1 mg/kg or 100 mg per dose
often require development of formulations at concentrations exceeding 100
mg/ml because of the small volume (<1.5 ml) that can be given by the
SC routes. For proteins that have a propensity to aggregate at the higher
concentrations, achieving such high concentration formulations is a
developmental challenge. Even for the IV delivery route, where large
volumes can be administered, protein concentrations of tens of milligrams
per milliliter may be needed for high dosing regimens and this may pose
stability challenges for some proteins.

[0006] The principles governing protein solubility are more complicated
than those for small synthetic molecules, and thus overcoming the protein
solubility issue takes different strategies. Operationally, solubility
for proteins could be described by the maximum amount of protein in the
presence of co-solutes whereby the solution remains visibly clear (i.e.,
does not show protein precipitates, crystals, or gels). The dependence of
protein solubility on ionic strength, salt form, pH, temperature, and
certain excipients has been mechanistically explained by changes in bulk
water surface tension and protein binding to water and ions versus
self-association by Arakawa et al in Theory of protein solubility,
Methods of Enzymology, 114:49-77, 1985; Schein in Solubility as a
function of protein structure and solvent components, BioTechnology
8(4):308-317, 1990; Jenkins in Three solutions of the protein solubility
problem, Protein Science 7(2):376-382, 1998; and others. Binding of
proteins to specific excipients or salts influences solubility through
changes in protein conformation or masking of certain amino acids
involved in self-interaction. Proteins are also preferentially hydrated
(and stabilized as more compact conformations) by certain salts, amino
acids, and sugars, leading to their altered solubility.

[0007] Aggregation which requires bi-molecular collision is expected to be
the primary degradation pathway in protein solutions. The relationship of
concentration to aggregate formation depends on the size of aggregates as
well as the mechanism of association. Protein aggregation may result in
covalent (e.g., disulfide-linked) or non-covalent (reversible or
irreversible) association. Irreversible aggregation by non-covalent
association generally occurs via hydrophobic regions exposed by thermal,
mechanical, or chemical processes that alter a protein's native
conformation. Protein aggregation may impact protein activity,
pharmacokinetics and safety, e.g., due to immunogenicity.

[0008] A typical approach to minimize aggregation, is to restrict the
mobility of proteins in order to reduce the number of collisions.
Lyophilization with appropriate excipients may improve protein stability
against aggregation by decreasing protein mobility and by restricting
conformational flexibility with the added benefit of minimizing
hydrolytic reactions consequent to removal of water. The addition of
appropriate excipients, including lyoprotectants, can prevent the
formation of aggregates during the lyophilization process as well as
during storage of the final product. A key parameter for effective
protection is the molar ratio of the lyoprotectant to the protein.
Generally molar ratios of 300:1 or greater are required to provide
suitable stability, especially for room temperature storage. Such ratios
can also, however, lead to an undesirable increase in viscosity.

[0009] Lyophilization allows for designing a formulation with appropriate
stability and tonicity. Although isotonicity is not necessarily required
for SC administration, it may be desirable for minimizing pain upon
administration. Isotonicity of a lyophile is difficult to achieve because
both the protein and the excipients are concentrated during the
reconstitution process. Excipient:protein molar ratios of 500:1 will
result in hypertonic preparations if the final protein concentration is
targeted for >100 mg/ml. If the desire is to achieve an isotonic
formulation, then a choice of lower molar ratio of excipient:protein will
result in a potentially less stable formulation.

[0010] Determining the highest protein concentration achievable remains an
empirical exercise due to the labile nature of protein conformation and
the propensity to interact with itself, with surfaces, and with specific
solutes.

[0011] Examples of subjects who may benefit from SC formulations are those
that have conditions that require frequent and chronic administration
such as subjects with the immune system disease rheumatoid arthritis and
immune disorders associated with graft transplantation. Commercially
available protein drug products for the treatment of rheumatoid arthritis
include HUMIRA®, ENBREL® and REMICADE®.

[0018] CTLA4Ig molecules interfere with T cell costimulation by inhibiting
the CD28-B7 interaction. Therefore, CTLA4Ig molecules can provide a
therapeutic use for immune system diseases, such as rheumatoid arthritis
and immune disorders associated with graft transplantation.

[0019] There is a need for a stable, effective convenient formulations
comprising CTLA4Ig molecules for treatment of immune system disorders.

[0021] The lyophilized formulation of the invention comprises the CTLA4Ig
molecule in a weight ratio of at least 1:2 protein to lyoprotectant. The
lyoprotectant is preferably sugar, more preferably disaccharides, most
preferably sucrose or maltose. The lyophilized formulation may also
comprise one or more of the components selected from the list consisting
of buffering agents, surfactants, bulking agents and preservatives.

[0022] In certain embodiments, the amount of sucrose or maltose useful for
stabilization of the lyophilized drug product is in a weight ratio of at
least 1:2 protein to sucrose or maltose, preferably in a weight ratio of
from 1:2 to 1:5 protein to sucrose or maltose, more preferably in a
weight ratio of about 1:2 protein to maltose or sucrose.

[0023] The subcutaneous (SC) formulation of the invention comprises the
CTLA4Ig molecule at a protein concentration of at least 100 mg/ml in
combination with a sugar at stabilizing levels, preferably a protein
concentration of at least 125 mg/ml in combination with a sugar at
stabilizing levels, in an aqueous carrier. The sugar is preferably in a
weight ratio of at least 1:1.1 protein to sugar. The stabilizer is
preferably employed in an amount no greater than that which may result in
a viscosity undesirable or unsuitable for administration via SC syringe.
The sugar is preferably disaccharides, most preferably sucrose. The SC
formulation may also comprise one or more of the components selected from
the list consisting of buffering agents, surfactants, and preservatives.

[0024] In certain embodiments, the amount of sucrose useful for
stabilization of the CTLA4Ig molecule SC drug product is in a weight
ratio of at least 1:1 protein to sucrose, preferably in a weight ratio of
from 1:1.3 to 1:5 protein to sucrose, more preferably in a weight ratio
of about 1:1.4 protein to sucrose.

[0025] The liquid formulation of the invention comprises the CTLA4Ig
molecule at a protein concentration of at least 20 mg/ml in combination
with a sugar at stabilizing levels, preferably at least 25 mg/ml in
combination with a sugar at stabilizing level in an aqueous carrier.
Preferably the sugar is in a weight ratio of at least 1:1 protein to
sugar. The sugar is preferably disaccharides, most preferably sucrose.
The liquid formulation may also comprise one or more of the components
selected from the list consisting of buffering agents, surfactants, and
preservatives.

[0026] In certain embodiments, the amount of sucrose useful for
stabilization of the liquid drug product is in a weight ratio of at least
1:1 protein to sucrose, preferably in a weight ratio of from 1:2 to 1:10
protein to sucrose, more preferably in a weight ratio of about 1:2
protein to sucrose.

[0027] In another embodiment of the invention, an article of manufacture
is provided which contains the drug product and preferably provides
instructions for its use.

[0028] The present invention further provides methods for treating immune
system diseases and tolerance induction by administering the CTLA4Ig
molecule formulations of the invention.

[0030] FIG. 2 depicts a nucleotide (SEQ ID NO:3) and amino acid (SEQ ID
NO:4) sequence of CTLA4-L104EA29Y-Ig (also know as "L104EA29YIg" and
"LEA29Y") comprising a signal peptide; a mutated extracellular domain of
CTLA4 starting at methionine at position +1 and ending at aspartic acid
at position +124, or starting at alanine at position -1 and ending at
aspartic acid at position +124; and an Ig region. SEQ ID NO: 3 and 4
depict a nucleotide and amino acid sequence, respectively, of L104EA29YIg
comprising a signal peptide; a mutated extracellular domain of CTLA4
starting at methionine at position +27 and ending at aspartic acid at
position +150, or starting at alanine at position +26 and ending at
aspartic acid at position +150; and an Ig region. L104EA29YIg can have
the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:4, (ii)
26-382 of SEQ ID NO:4; (iii) 27-383 of SEQ ID NO:4 or (iv) 27-382 of SEQ
ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ
ID NO:4.

DETAILED DESCRIPTION OF THE INVENTION

[0031] As utilized herein:

[0032] A "stable" formulation or drug product is one in which the CTLA4Ig
molecule therein essentially retains its physical and chemical stability
and integrity upon storage. Stability of the CTLA4Ig molecule
formulations can be measured at selected temperatures after selected time
periods. For example, an increase in aggregate formation following
lyophilization and storage is an indicator for instability of a
lyophilized. CTLA4Ig molecule formulation. In addition to aggregate
formation, retention of original clarity, color and odor throughout shelf
life are indicators utilized to monitor stability of CTLA4Ig molecule
solutions. HMW species are multimers (i.e. tetramers, hexamers, etc),
which have a higher molecular weight than CTLA4Ig molecule dimers.
Typically a "stable" drug product may be one wherein the increase in
aggregation, as measured by an increase in the percentage of high
molecular weight species (% HMW), is less than about 5% and preferably
less than about 3%, when the formulation is stored at 2-8° C. for
one year. Preferably, the manufactured drug product comprises less than
about 25% HMW species, preferably less than about 15% HMW species, more
preferably less than about 10% HMW species, most preferred less than
about 5% HMW species.

[0033] The monomer, dimer and HMW species of CTLA4Ig molecule may be
separated by size exclusion chromatography (SEC). SEC separates molecules
based on the molecular size. Separation is achieved by the differential
molecular exclusion or inclusion as the molecules migrate along the
length of the column. Thus, resolution increases as a function of column
length. CTLA4Ig molecule samples may be separated using a 2695 Alliance
HPLC (Waters, Milford, Mass.) equipped with TSK Gel® G3000SWXL (300
mm×7.8 mm) and TSK Gel® G3000SWXL (40 mm×6.0 mm) columns
(Tosoh Bioscience, Montgomery, Pa.) in tandem. Samples at 10 mg/ml (20
μl aliquot) are separated using a mobile phase consisting of 0.2 M
KH2PO4, 0.9% NaCl, pH 6.8, at a flow rate of 1.0 ml/min.
Samples are monitored at an absorbance of 280 nm using Water's 2487 Dual
Wavelength detector. Using this system, the HMW species has a retention
time of 7.5 min±1.0 min. Each peak is integrated for area under the
peak. The % HMW species calculated by dividing the HMW peak area by the
total peak area.

[0034] A "reconstituted" formulation is one which has been prepared by
dissolving a lyophilized formulation in an aqueous carrier such that the
CTLA4Ig molecule is dissolved in the reconstituted formulation. The
reconstituted formulation is suitable for intravenous administration (IV)
to a patient in need thereof.

[0035] An "isotonic" formulation is one which has essentially the same
osmotic pressure as human blood. Isotonic formulations will generally
have an osmotic pressure from about 250 to 350 mOsmol/KgH2O. The term
"hypertonic" is used to describe a formulation with an osmotic pressure
above that of human blood. Isotoni city can be measured using a vapor
pressure or ice-freezing type osmometer, for example.

[0036] The term "buffering agent" refers to one or more components that
when added to an aqueous solution is able to protect the solution against
variations in pH when adding acid or alkali, or upon dilution with a
solvent. In addition to phosphate buffers, there can be used glycinate,
carbonate, citrate buffers and the like, in which case, sodium, potassium
or ammonium ions can serve as counterion.

[0037] An "acid" is a substance that yields hydrogen ions in aqueous
solution. A "pharmaceutically acceptable acid" includes inorganic and
organic acids which are non toxic at the concentration and manner in
which they are formulated.

[0038] A "base" is a substance that yields hydroxyl ions in aqueous
solution. "Pharmaceutically acceptable bases" include inorganic and
organic bases which are non-toxic at the concentration and manner in
which they are formulated.

[0039] A "lyoprotectant" is a molecule which, when combined with a protein
of interest, prevents or reduces chemical and/or physical instability of
the protein upon lyophilization and subsequent storage.

[0040] A "preservative" is an agent that reduces bacterial action and may
be optionally added to the formulations herein. The addition of a
preservative may, for example, facilitate the production of a multi-use
(multiple-dose) formulation. Examples of potential preservatives include
octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride,
benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides
in which the alkyl groups are long-chain compounds), and benzethonium
chloride. Other types of preservatives include aromatic alcohols such as
phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl
paraben, catechol, resorcinol, cyclohexanol, 3pentanol, and m-cresol.

[0042] A "drug substance" refers to the starting material utilized in
formulation of the final drug product. Typical CTLA4Ig drug substance
composition comprises a protein concentration from 20 mg/ml to 60 mg/ml,
pH from 7 to 8 and % HMW species of <5%.

[0043] A "formulated bulk solution" refers to the final formulation prior
to filling of the container such as the formulated solution prior to
filling the vials for lyophilization, or the formulated solution prior to
filling the syringe for SC injection.

[0044] A "drug product" refers to the final formulation packaged in a
container which may be reconstituted before use, such as with a
lyophilized drug product; diluted further before use, such as with a
liquid drug product; or utilized as is, such as with a SC solution drug
product.

[0045] The terms "CTLA4-Ig" or "CTLA4-Ig molecule" or "CTLA4Ig molecule"
are used interchangeably, and refer to a protein molecule that comprises
at least a polypeptide having a CTLA4 extracellular domain or portion
thereof and an immunoglobulin constant region or portion thereof. The
extracellular domain and the immunoglobulin constant region can be
wild-type, or mutant or modified, and mammalian, including human or
mouse. The polypeptide can further comprise additional protein domains. A
CTLA4-Ig molecule can also refer to multimer forms of the polypeptide,
such as dimers, tetramers, and hexamers. A CTLA4-Ig molecule also is
capable of binding to CD80 and/or CD86.

[0046] The term "B7-1" refers to CD80; the term "B7-2" refers CD86; and
the term "B7" refers to both B7-1 and B7-2 (CD80 and CD86). The term
"B7-1-Ig" or "B7-1Ig" refers to CD80-Ig; the term "B7-2-Ig" or "B7-2Ig"
refers CD86-Ig.

[0047] In one embodiment, "CTLA4Ig" refers to a protein molecule having
the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2, (ii)
26-382 of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ
ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ
ID NO:2. In monomeric form these proteins can be referred to herein as
"SEQ ID NO:2 monomers," or monomers "having a SEQ ID NO:2 sequence".
These SEQ ID NO:2 monomers can dimerize, such that dimer combinations can
include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and
(iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and
(iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii)
and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v)
and (v); (v) and (vi); and, (vi) and (vi). These different dimer
combinations can also associate with each other to form tetramer CTLA4Ig
molecules. These monomers, dimers, tetramers and other multimers can be
referred to herein as "SEQ ID NO:2 proteins" or proteins "having a SEQ ID
NO:2 sequence". (DNA encoding CTLA4Ig as shown in SEQ ID NO:2 was
deposited on May 31, 1991 with the American Type Culture Collection
(ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 under the
provisions of the Budapest Treaty, and has been accorded ATCC accession
number ATCC 68629; a Chinese Hamster Ovary (CHO) cell line expressing
CTLA4Ig as shown in SEQ ID NO:2 was deposited on May 31, 1991 with ATCC
identification number CRL-10762). As utilized herein "Abatacept" refers
to SEQ ID NO:2 proteins.

[0048] In one embodiment, CTLA4-L104EA29Y-Ig (sometimes known as "LEA29Y"
or "L104EA29Y") is a genetically engineered fusion protein similar in
structure to CTAL4-Ig molecule as shown in SEQ ID NO: 1. L104EA29Y-Ig has
the functional extracellular binding domain of modified human CTLA4 and
the Fc domain of human immunoglobulin of the IgG1 class. Two amino acid
modifications, leucine to glutamic acid at position 104 (L104E), which is
position 130 of SEQ ID NO:2, and alanine to tyrosine at position 29
(A29Y), which is position 55 of SEQ ID NO:2, were made in the B7 binding
region of the CTLA4 domain to generate L104EA29Y. SEQ ID NOS: 3 and 4
depict a nucleotide and amino acid sequence, respectively, of L104EA29YIg
comprising a signal peptide; a mutated extracellular domain of CTLA4
starting at methionine at position +27 and ending at aspartic acid at
position +150, or starting at alanine at position +26 and ending at
aspartic acid at position +150; and an Ig region. DNA encoding
L104EA29Y-Ig was deposited on Jun. 20, 2000, with the American Type
Culture Collection (ATCC) under the provisions of the Budapest Treaty. It
has been accorded ATCC accession number PTA-2104. L104EA29Y-Ig is farther
described in U.S. Pat. No. 7,094,874, issued on Aug. 22, 2006, and in WO
01/923337 A2, which are incorporated by reference herein in their
entireties.

[0049] Expression of L104EA29YIg in mammalian cells can result in the
production of N- and C-terminal variants, such that the proteins produced
can have the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:4,
(ii) 26-382 of SEQ ID NO:4; (iii) 27-383 of SEQ ID NO:4 or (iv) 27-382 of
SEQ ID NO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of
SEQ ID NO:4. In monomeric form these proteins can be referred to herein
as "SEQ ID NO:4 monomers," or monomers "having a SEQ ID NO:4 sequence.
These proteins can dimerize, such that dimer combinations can include,
for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i)
and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii)
and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v);
(iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v);
(v) and (vi); and, (vi) and (vi)." These different dimer combinations can
also associate with each other to form tetramer L104EA29YIg molecules.
These monomers, dimers, tetramers and other multimers can be referred to
herein as "SEQ ID NO:4 proteins" or proteins "having a SEQ ID NO:4
sequence". As utilized herein "Belatacept" refers to SEQ ID NO:4
proteins.

CTLA4-Ig Monomers and Multimers

[0050] CTLA4-Ig molecules can include, for example, CTLA4-Ig proteins in
monomer, dimer, trimer, tetramer, pentamer, hexamer, or other multimeric
forms. CTLA4-Ig molecules can comprise a protein fusion with at least an
extracellular domain of CTLA4 and an immunoglobulin constant region.
CTLA4-Ig molecules can have wild-type or mutant sequences, for example,
with respect to the CTLA4 extracellular domain and immunoglobulin
constant region sequences. CTLA4-Ig monomers, alone, or in dimer,
tetramer or other multimer form, can be glycosytated.

[0051] In some embodiments, the invention provides populations of CTLA4-Ig
molecules that have at least a certain percentage of dimer or other
multimer molecules. For example, the invention provides CTLA4-Ig molecule
populations that are greater than 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%
CTLA4-Ig dimers. In one embodiment, the invention provides a CTLA4-Ig
molecule population that comprises from about 95% to about 99.5% CTLA4-Ig
dimer and from about 0.5% to about 5% of CTLA4-Ig tetramer. In another
embodiment, the CTLA4-Ig molecule population comprises about 98% CTLA4-Ig
dimer, about 1.5% CTLA4-Ig tetramer and about 0.5% CTLA4-Ig monomer.

[0052] In one embodiment, the invention provides a population of CTLA4-Ig
molecules wherein the population is substantially free of CTLA4-Ig
monomer molecules. Substantially free of CTLA4-Ig monomer molecules can
refer to a population of CTLA4-Ig molecules that have less than 1%, 0.5%,
or 0.1% of monomers.

[0053] In one embodiment, the invention provides a population of CTLA4-Ig
molecules wherein the population is substantially free of CTLA4-Ig
multimers that are larger than dimers, such as tetramers, hexamers, etc.
Substantially free of CTLA4-Ig multimer molecules larger than dimers can
refer to a population of CTLA4-Ig molecules that have less than 6%, 5%,
4%, 3%, 2%, 1%, 0.5%, or 0.1% of CTLA4-Ig multimers larger than dimeric
form.

[0054] In one embodiment, a CTLA4-Ig monomer molecule can have, for
example, the amino acid sequence of: (i) 26-383 of SEQ ID NO:2, (ii)
26-382 of SEQ ID NO:2 (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ
ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ
ID NO:2. When an expression cassette comprising the nucleic acid sequence
of SEQ ID NO: 1 is expressed in CHO cells, the predominant monomer form
expressed has the N-terminus amino acid residue of methionine (residue 27
of SEQ ID NO:2), which corresponds to the N-terminus amino acid residue
of wild-type human CTLA4. However, because SEQ ID NO:1 also includes the
coding sequence for an Oncostatin M Signal Sequence (nucleotides 11-88 of
SEQ ID NO: 1), the expressed protein from SEQ ID NO:1 contains an
Oncostatin M Signal Sequence. The signal sequence is cleaved from the
expressed protein during the process of protein export from the
cytoplasm, or secretion out of the cell. But cleavage can result in
N-terminal variants, such as cleavage between amino acid residues 25 and
26 (resulting in an N-terminus of residue 26, i.e., the "Ala variant"),
or between amino acid residues 24 and 25 (resulting in an N-terminus of
residue 2, i.e., the "Met-Ala variant"), as opposed to cleavage between
amino acid residues 26 and 27 (resulting in an N-terminus of residue 27).
For example, the Met-Ala variant can be present in a mixture of CTLA4-Ig
molecules at about 1%, and the Ala variant can be present in a mixture of
CTLA4-Ig molecules at about 8-10%. In addition, the expressed protein
from SEQ ID NO:1 can have C-terminus variants due to incomplete
processing. The predominant C-terminus is the glycine at residue 382 of
SEQ ID NO:2. In a mixture of CTLA4-Ig molecules, monomers having lysine
at the C-terminus (residue 383 of SEQ ID NO:2) can be present, for
example, at about 4-5%.

[0055] A CTLA4-Ig monomer molecule can comprise an extracellular domain of
human CTLA4. In one embodiment, the extracellular domain can comprise the
nucleotide sequence of nucleotides 89-463 of SEQ ID NO:1 that code for
amino acids 27-151 of SEQ ID NO:2. In another embodiment, the
extracellular domain can comprise mutant sequences of human CTLA4. In
another embodiment, the extracellular domain can comprise nucleotide
changes to nucleotides 89-463 of SEQ ID NO:1 such that conservative amino
acid changes are made. In another embodiment, the extracellular domain
can comprise a nucleotide sequence that is at least 75%, 80%, 85%, 90%,
95%, 96%, 97%, 98%, or 99% identical to nucleotides 89-463 of SEQ ID
NO:1.

[0056] A CTLA4-Ig monomer molecule can comprise a constant region of a
human immunoglobulin. This constant region can be a portion of a constant
region; this constant region can have a wild-type or mutant sequence. The
constant region can be from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1,
IgA2, IgD or IgE. The constant region can be from a light chain or a
heavy chain of an immunoglobulin. Where the constant region is from an
IgG, IgD, or IgA molecule, the constant region can comprise one or more
of the following constant region domains: CL, CH1, hinge, CH2, or CH3.
Where the constant region is from IgM or IgE, the constant region can
comprise one or more of the following constant region domains: CL, CH1,
CH2, CH3, or Ca4. In one embodiment, the constant region can comprise on
or more constant region domains from IgG, IgD, IgA, IgM or IgE.

[0059] In one embodiment, a CTLA4-Ig molecule population comprises
monomers having a sequence shown in any one or more of FIG. 7, 8, or 9 of
the U.S. Pat. No. 7,094,874, issued on Aug. 22, 2006 and in U.S. patent
applications published as Publication No. US20030083246 and
US20040022787, which are hereby incorporated by reference in its
entirety.

[0060] In one embodiment, a CTLA4-Ig tetramer molecule comprises two pairs
or two dimers of CTLA4-Ig polypeptides, wherein each polypeptide has one
of the following amino acid sequences: (i) 26-383 of SEQ ID NO:2, (ii)
26-382 of SEQ ID NO:2, (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382 of SEQ
ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ
ID NO:2. Each member of the pair of polypeptides or dimer is covalently
linked to the other member, and the two pairs of polypeptides are
non-covalently associated with one another thereby forming a tetramer.
Such tetramer molecules are capable of binding to CD80 or CD86.

[0061] In another embodiment, such tetramer molecules can bind to CD80 or
CD86 with an avidity that is at least 2-fold greater than the binding
avidity of a CTLA4-Ig dimer (whose monomers have one of the above amino
acid sequences) to CD80 or CD86. In another embodiment, such tetramer
molecules can bind to CD80 or CD86 with an avidity that is at least
2-fold greater than the binding affinity or avidity of wild-type CTLA4 to
CD80 or CD86. Such greater avidity can contribute to higher efficacy in
treating immune disorders and other diseases as described below. In
addition, greater or improved avidity can produce the result of higher
potency of a drug. For example, a therapeutic composition comprising
CTLA4-Ig tetramer would have a higher avidity and therefore higher
potency than the same amount of a therapeutic composition having CTLA4-Ig
monomer. In another embodiment, such tetramer molecules can have at least
a 2-fold greater inhibition on T cell proliferation as compared to a
CTLA4-Ig dimer (whose monomers have one of the above amino acid
sequences). In another embodiment, such tetramer molecules can have at
least a 2-fold greater inhibition on T cell proliferation as compared to
a wild-type CTLA4 molecule.

[0062] T cell proliferation can be measured using standard assays known in
the art. For example, one of the most common ways to assess T cell
proliferation is to stimulate T cells via antigen or agonistic antibodies
to TCR and to measure, for example, the incorporation of titrated
thymidine (3H-TdR) in proliferating T cells or the amount of cytokines
released by proliferating T cells into culture. The inhibitory effect of
CTLA4-Ig molecules upon T cell activation or proliferation can thereby be
measured.

[0063] The affinity of a CTLA4-Ig molecule is the strength of binding of
the molecule to a single ligand, including CD80, CD86, or CD8OIg or
CD86Ig fusion proteins. The affinity of CTLA4-Ig to ligands can be
measured by using, for example, binding interaction analysis (BIA) based
on surface plasmon technique. Aside from measuring binding strength, it
permits real time determination of binding kinetics, such as association
and dissociation rate constants. A sensor chip, consisting of a glass
slide coated with a thin metal film, to which a surface matrix is
covalently attached, is coated with one of the interactants, i.e,
CTLA4-Ig or one of the ligands. A solution containing the other
interactant is allowed to flow over its surface. A continuous light beam
is directed against the other side of the surface, and its reflection
angle is measured. On binding of CTLA4-Ig to the ligand, the resonance
angle of the light beam changes (as it depends on the refractive index of
the medium close to the reactive side of the sensor, which in turn is
directly correlated to the concentration of dissolved material in the
medium). It is subsequently analyzed with the aid of a computer.

[0064] In one embodiment, CTLA4-Ig binding experiments can be performed by
surface plasmon resonance (SPR) on a BIAcore instrument (BIAcore AG,
Uppsala, Sweden). CTLA4-Ig can be covalently coupled by primary amine
groups to a carboxymethylated dextran matrix on a BIAcore sensor chip,
thereby immobilizing CTLA4-Ig to the sensor chip. Alternatively, an
anti-constant region antibody can be used to immobilize CTLA4-Ig
indirectly to the sensor surface via the Ig fragment. Thereafter, ligands
are added to the chip to measure CTLA4-Ig binding to the ligands.
Affinity measurements can be performed, for example, as described in van
der Merwe, P. et al., J. Exp. Med. (1997) 185 (3):393-404.

[0065] The avidity of CTLA4-Ig molecules can also be measured. Avidity can
be defines as the sum total of the strength of binding of two molecules
or cells to one another at multiple sites. Avidity is distinct from
affininty which is the strength of binding one site on a molecule to its
ligand. Without being bound by theory, higher avidity of CTLA4-Ig
molecules can lead to increased potency of inhibition by CTLA4-Ig
molecules on T-cell proliferation and activation. Avidity can be
measured, for example, by two categories of solid phase assays: a)
competitive inhibition assays, and b) elution assays. In both of them the
ligand is attached to a solid support. In the competitive inhibition
assay, CTLA4-Ig molecules are then added in solution at a fixed
concentration, together with free ligand in different concentrations, and
the amount of ligand which inhibits solid phase binding by 50% is
determined. The less ligand needed, the stronger the avidity. In elution
assays, the ligand is added in solution. After obtaining a state of
equilibrium, a chaotrope or denaturant agent (e.g. isothiocyanate, urea,
or diethylamine) is added in different concentrations to disrupt
CTLA4-Ig/ligand interactions. The amount of CTLA4-Ig resisting elution is
determined thereafter with an ELISA. The higher the avidity, the more
chaotropic agent is needed to elute a certain amount of CTLA4-Ig. The
relative avidity of a heterogeneous mixture of CTLA4-Ig molecules can be
expressed as the avidity index (AI), equal to the concentration of
eluting agent needed to elute 50% of the bound CTLA4-Ig molecules.
Refined analysis of data can be performed by determining percentages of
eluted CTLA4-Ig at different concentrations of the eluting agent.

Methods for Producing the CTLA4Ig Molecules of the Invention

[0066] Expression of CTLA4Ig molecules can be in prokaryotic cells.
Prokaryotes most frequently are represented by various strains of
bacteria. The bacteria may be a gram positive or a gram negative.
Typically, gram-negative bacteria such as E. coli are preferred. Other
microbial strains may also be used.

[0067] Sequences, described above, encoding CTLA4Ig molecules can be
inserted into a vector designed for expressing foreign sequences in
prokaryotic cells such as E. coli. These vectors can include commonly
used prokaryotic control sequences which are defined herein to include
promoters for transcription initiation, optionally with an operator,
along with ribosome binding site sequences, include such commonly used
promoters as the beta-lactamase (penicillinase) and lactose (lac)
promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan
(trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived PL promoter and N-gene ribosome binding site
(Shimatake, et al., (1981) Nature 292:128).

[0068] Such expression vectors will also include origins of replication
and selectable markers, such as a beta-lactamase or neomycin
phosphotransferase gene conferring resistance to antibiotics, so that the
vectors can replicate in bacteria and cells carrying the plasmids can be
selected for when grown in the presence of antibiotics, such as
ampicillin or kanamycin.

[0071] Nucleic acid sequences encoding CTLA4Ig molecules described above
can also be inserted into a vector designed for expressing foreign
sequences in a eukaryotic host. The regulatory elements of the vector can
vary according to the particular eukaryotic host.

[0072] Commonly used eukaryotic control sequences for use in expression
vectors include promoters and control sequences compatible with mammalian
cells such as, for example, CMV promoter (CDM8 vector) and avian sarcoma
virus (ASV) (πLN vector). Other commonly used promoters include the
early and late promoters from Simian Virus 40 (SV40) (Fiers, et al.,
(1973) Nature 273:113), or other viral promoters such as those derived
from polyoma, Adenovirus 2, and bovine papilloma virus. An inducible
promoter, such as hMTII (Karin, et al., (1982) Nature 299:797-802) may
also be used.

[0073] Vectors for expressing CTLA4Ig molecules in eukaryotes may also
carry sequences called enhancer regions. These are important in
optimizing gene expression and are found either upstream or downstream of
the promoter region.

[0078] Other promoters are inducible because they can be regulated by
environmental stimuli or the growth medium of the cells. These inducible
promoters include those from the genes for heat shock proteins, alcohol
dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associated
with nitrogen catabolism, and enzymes responsible for maltose and
galactose utilization.

[0079] Regulatory sequences may also be placed at the 3' end of the coding
sequences. These sequences may act to stabilize messenger RNA. Such
terminators are found in the 3' untranslated region following the coding
sequences in several yeast-derived and mammalian genes.

[0081] Mammalian cells can be transformed by methods including but not
limited to, transfection in the presence of calcium phosphate,
microinjection, electroporation, or via transduction with viral vectors.

[0082] Methods for introducing foreign DNA sequences into plant and yeast
genomes include (1) mechanical methods, such as microinjection of DNA
into single cells or protoplasts, vortexing cells with glass beads in the
presence of DNA, or shooting DNA-coated tungsten or gold spheres into
cells or protoplasts; (2) introducing DNA by making cell membranes
permeable to macromolecules through polyethylene glycol treatment or
subjection to high voltage electrical pulses (electroporation); or (3)
the use of liposomes (containing cDNA) which fuse to cell membranes.

[0083] US patent application US Publication Number 20050019859 and US
patent application US Publication Number 20050084933 teach processes for
the production of proteins of the invention, specifically recombinant
glycoprotein products, by animal or mammalian cell cultures and are
herein incorporated by reference.

[0084] Following the protein production phase of the cell culture process,
CTLA4Ig molecules are recovered from the cell culture medium using
techniques understood by one skilled in the art. In particular, the
CTLA4Ig molecule is recovered from the culture medium as a secreted
polypeptide.

[0085] The culture medium is initially centrifuged to remove cellular
debris and particulates. The desired protein subsequently is purified
from contaminant DNA, soluble proteins, and polypeptides, with the
following non-limiting purification procedures well-established in the
art: SDS-PAGE; ammonium sulfate precipitation; ethanol precipitation;
fractionation on immunoaffinity or ion-exchange columns; reverse phase
HPLC; chromatography on silica or on an anion-exchange resin such as QAE
or DEAE; chromatofocusing; gel filtration using, for example, Sephadex
G-75 ® column; and protein A Sepharose® columns to remove
contaminants such as IgG. Addition of a protease inhibitor, such as
phenyl methyl sulfonyl fluoride (PMSF), or a protease inhibitor cocktail
mix also can be useful to inhibit proteolytic degradation during
purification. A person skilled in the art will recognize that
purification methods suitable for a protein of interest, for example a
glycoprotein, can require alterations to account for changes in the
character of the protein upon expression in recombinant cell culture.

[0086] Purification techniques and methods that select for the
carbohydrate groups of the glycoprotein are also of utility within the
context of the present invention. For example, such techniques include,
HPLC or ion-exchange chromatography using cation- or anion-exchange
resins, wherein the more basic or more acidic fraction is collected,
depending on which carbohydrate is being selected for. Use of such
techniques also can result in the concomitant removal of contaminants.

[0087] The purification method can farther comprise additional steps that
inactivate and/or remove viruses and/or retroviruses that might
potentially be present in the cell culture medium of mammalian cell
lines. A significant number of viral clearance steps are available,
including but not limited to, treating with chaotropes such as urea or
guanidine, detergents, additional ultrafiltration/diafiltration steps,
conventional separation, such as ion-exchange or size exclusion
chromatography, pH extremes, heat, proteases, organic solvents or any
combination thereof.

[0088] The purified CTLA4Ig molecule require concentration and a buffer
exchange prior to storage or further processing. A Pall Filtron TFF
system may be used to concentrate and exchange the elution buffer from
the previous purification column with the final buffer desired for the
drug substance.

[0089] In one aspect, purified CTLA4Ig molecules, which have been
concentrated and subjected to diafiltration step, can be filled into 2-L
Biotainer® bottles, 50-L bioprocess bag or any other suitable vessel.
CTLA4Ig molecules in such vessels can be stored for about 60 days at
2° to 8° C. prior to freezing. Extended storage of purified
CTLA4Ig molecules at 2° to 8° C. may lead to an increase in
the proportion of HMW species. Therefore, for long-term storage, CTLA4Ig
molecules can be frozen at about -70° C. prior to storage and
stored at a temperate of about -40° C. The freezing temperature
can vary from about -50° C. to about -90° C. The freezing
time can vary and largely depends on the volume of the vessel that
contains CTLA4Ig molecules, and the number of vessels that are loaded in
the freezer. For example, in one embodiment, CTLA4Ig molecules are in 2-L
Biotainer® bottles. Loading of less than four 2-L Biotainer®
bottles in the freezer may require from about 14 to at least 18 hours of
freezing time. Loading of at least four bottles may require from about 18
to at least 24 hours of freezing time. Vessels with frozen CTLA4Ig
molecules are stored at a temperature from about -35° C. to about
-55° C. The storage time at a temperature of about -35° C.
to about -55° C. can vary and can be as short as 18 hours. The
frozen drug substance can be thawed in a control manner for formulation
of drug product.

[0091] The lyophilized formulation of the invention comprises the CTLA4Ig
molecule in a weight ratio of at least 1:2 protein to lyoprotectant. The
lyoprotectant is preferably sugar, more preferably disaccharides, most
preferably sucrose or maltose. The lyophilized formulation may also
comprise one or more of the components selected from the list consisting
of buffering agents, surfactants, bulking agents and preservatives.

[0092] During formulation development studies, the effects of various
excipients on the solution- and freeze-dried solid-state stability of the
CTLA4Ig molecule were evaluated.

[0093] Instability of the freeze-dried CTLA4Ig molecule in the absence of
a stabilizer clearly highlighted a need for inclusion of a lyoprotectant
in the formulation. Initial screening studies showed that drug product
was stable in the presence of sugars such as maltose and sucrose and
amino acids such as arginine and lysine. Polyols such as mannitol and
polysaccharides such as dextran 40 were detrimental to its stability. The
preferred lyoprotectants are the disaccharides maltose and sucrose.

[0094] Example VI describes stability studies of freeze dried Abatacept
drug product in the presence of maltose stored at 50° C. An
increase in high molecular weight (HMW) species was monitored using a
stability-indicating size exclusion chromatography (SE-HPLC) assay. The
results demonstrate that the stability of the CTLA4Ig molecule in a
freeze-dried solid-state form is enhanced in the presence of maltose.

[0095] The amount of sucrose or maltose useful for stabilization of the
lyophilized drug product is in a weight ratio of at least 1:2 protein to
sucrose or maltose, preferably in a weight ratio of from 1:2 to 1:5
protein to sucrose or maltose, more preferably in a weight ratio of about
1:2 protein to maltose or sucrose.

[0096] If necessary, the pH of the formulation, prior to lyophilization,
is set by addition of a pharmaceutically acceptable acid and/or base. The
preferred pharmaceutically acceptable acid is hydrochloric acid. The
preferred base is sodium hydroxide.

[0097] During formulation development, the stability of the freeze-dried
drug product was studied as a function of pH. Example VI describes
stability studies with freeze dried Abatacept drug product as a function
of pH. The solution pH was adjusted between 6 to 8 prior to
freeze-drying. The samples were placed on stability and the constituted
product vials were monitored for an increase in the high molecular weight
species at various time points using a stability-indicating
size-exclusion chromatography (SE-HPLC) assay. Under the recommended
storage condition of 2°-8° C., no significant changes in
the rate of formation of HMW species were observed. Additionally; the
solution-state stability data generated during an early development
showed the pH of maximum stability to be between 7 and 8. The acceptable
pH range for the lyophilized drug product is from 7 to 8 with a preferred
target pH of 7.5.

[0098] In another aspect, the salts or buffer components may be added in
an amount of at least about 10 mM, preferably 10-200 mM. The salts and/or
buffers are pharmaceutically acceptable and are derived from various
known acids (inorganic and organic) with "base forming" metals or amines.
In addition to phosphate buffers, there can be used glycinate, carbonate,
citrate buffers and the like, in which case, sodium, potassium or
ammonium ions can serve as counterion.

[0099] A "bulking agent" is a compound which adds mass to a lyophilized
mixture and contributes to the physical structure of the lyophilized cake
(e.g. facilitates the production of an essentially uniform lyophilized
cake which maintains an open pore structure). Illustrative bulking agents
include mannitol, glycine, polyethylene glycol and sorbitol. The
lyophilized formulations of the present invention may contain such
bulking agents

[0100] A preservative may be optionally added to the formulations herein
to reduce bacterial action. The addition of a preservative may, for
example, facilitate the production of a multi-use (multiple-dose)
formulation.

[0101] One skilled in the art would select the amount of drug product to
be filed into a vial depending on the required dosages and administration
schedule for a specific treatment. For example, the concentration of
CTLA4Ig per vial may range from 50 to 300 mg/vial, preferably 100 to 250
mg/vial.

[0104] The lyophilized drug product is constituted with an aqueous
carrier. The aqueous carrier of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration to a
human) and is useful for the preparation of a liquid formulation, after
lyophilization. Illustrative diluents include sterile water for injection
(SWFI), bacteriostatic water for injection (BWFI), a pH buffered solution
(e.g. phosphate-buffered saline), sterile saline solution, Ringer's
solution or dextrose solution.

[0105] Preferably, the lyophilized drug product of the current invention
is constituted with either Sterile Water for Injection, USP (SWFI) or
0.9% Sodium Chloride Injection, USP. During constitution, the lyophilized
powder rapidly dissolves providing a clear, colorless solution.

[0106] Typically, the lyophilized drug product of the instant invention is
constituted to about 25 mg/ml with 10 ml of either Sterile Water for
injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. The
constituted solution is further diluted to drug product concentrations
between 1 and 10 mg/ml with 0.9% Sodium Chloride Injection, USP. The
diluted drug product for injection is isotonic and suitable for
administration by intravenous infusion.

[0107] During early clinical development, the constituted solutions of
lyophilized drug product were found to be incompatible with disposable
siliconized syringes, which are commonly used for the preparation and
administration of the parenteral products. Specifically, the constituted
drug product solution developed thread-like, gelatinous particles when
stored in these syringes for more than 10-15 minutes. Further studies
demonstrated that this incompatibility was due to the interaction of drug
product with silicone oil (dimethylsiloxane), which is used as a
lubricant on these syringes. The formation of these particles did not
affect the potency of the drug product solution over its use time.
Silicone free syringes are preferably utilized for drug product
constitution and transfer of the constituted solutions from the vial to
the intravenous bag.

[0108] Alternatively, a surfactant may be added to the formulation to
reduce or prevent the interaction of the constituted drug product with
the siliconized syringe, for example, in an amount sufficient thereof.

[0109] The recommended storage condition for the lyophilized formulation
is from 2-8° C. with a recommended shelf life of at least 12
months.

Preparation of the Lyophilized Formulation

[0110] The lyophilized drug product manufacturing process involves
batching of the Formulated Bulk Solution for lyophilization, aseptic
filtration, filling in vials, freezing vials in a freeze-dryer chamber,
followed by lyophilization, stoppering and capping.

[0112] The Formulated Bulk Solution is typically set at a fixed protein
concentration so that the desired vial fill volume can be kept constant.
During the addition of lyoprotectant, the mixer speed is controlled at
250±50 rpm so as to minimize foaming in the batching solution and to
ensure complete dissolution of the lyoprotectant within 10 to 20 minutes.
The Formulated Bulk Solution can be stored under 2°-8° C.
or room temperature and room light for at least 32 hours prior to
lyophilization.

[0113] The Formulated Bulk Solution is not terminally sterilized due to
heat sensitivity of the CTLA4Ig molecule. The Formulated Bulk Solution
may be sterilized using two 0.22 μm Millipore Millipak®
sterilizing grade filters in series prior to filling in vials for
lyophilization.

[0114] The freeze-drying cycle selected for this product is optimized in
order to have efficient sublimation and evaporation during the primary
and secondary phases of drying respectively, without compromising the
product quality.

[0115] To aid in rapid dissolution of the lyophilized powder and to
prevent the formation of excessive foam during constitution of the drug
product, the lyophilized vials are stoppered under 500±100 microns
chamber pressure at the end of the freeze-drying cycle.

[0116] One skilled in the art would be aware of the need to overfill the
container so as to compensate for vial, needle, syringe hold-up during
preparation and injection. For example, each vial of Abatacept drug
product, 250 mg/ml, contains a 5% overage of drug product to account for
reconstitution and withdrawal losses.

Liquid Subcutaneous Formulation

[0117] One skilled in the art would recognize the inconvenience of an IV
formulation for the patient in need of frequent, chronic therapy. The
patient has to make frequent trips to the hospital to receive their drug
via an IV infusion that may last as long as an hour. Consequently, a SC
formulation that could be self-administered at home would be very
beneficial to such a patient.

[0118] For subcutaneous administration, a dosage form with high protein
concentrations is desired. Treatments with high doses of more than 1
mg/kg (>100 mg per dose) require development of formulations at
concentrations exceeding 100 mg/ml because of the small volume (<1.5
ml) that can be given by the SC routes. In order to optimize the long
term stability at high concentration against formation of high molecular
weight species, formulation development studies were conducted to
evaluate the effect of various excipients on the solution state stability
of the liquid SC formulations of the invention.

[0119] The SC formulation of the invention comprises the CTLA4Ig molecule
at a protein concentration of at least 100 mg/ml in combination with a
sugar at stabilizing levels, preferably a protein concentration of at
least 125 mg/ml in combination with a sugar at stabilizing levels, in an
aqueous carrier. The sugar is preferably in a weight ratio of at least
1:1.1 protein to sugar. The stabilizer is preferably employed in an
amount no greater than that which may result in a viscosity undesirable
or unsuitable for administration via SC syringe. The sugar is preferably
disaccharides, most preferably sucrose. The SC formulation may also
comprise one or more of the components selected from the list consisting
of buffering agents, surfactants, and preservatives.

[0120] The stability profile of the SC formulation was evaluated in
presence of various stabilizers including sugars, polysaccharides, amino
acids, surfactants, polymers, cyclodextrans, proteins, etc. Among all the
excipients evaluated, sugars such as sucrose, mannitol and trehalose had
a better stabilizing effect against the formation of high molecular
weight species.

[0121] Examples V and VIII describe stability studies of SC Belatacept and
Abatacept drug product, respectively, in the presence of sucrose stored
at various temperatures for various time periods. An increase in high
molecular weight species was monitored using a stability-indicating size
exclusion chromatography (SE-HPLC) assay. The results demonstrate that
the stability of the CTLA4Ig molecule in the SC formulation is enhanced
in the presence of sucrose. Stabilization by sucrose was better at higher
sucrose:protein weight ratio. Based on these studies, sucrose was
selected as the stabilizer at a ratio that provides optimum stability
without resulting in a SC solution with excessive hypertonicity.

[0122] The amount of sucrose useful for stabilization of the SC drug
product is in a weight ratio of at least 1:1 protein to sucrose,
preferably in a weight ratio of from 1:1.3 to 1:5 protein to sucrose,
more preferably in a weight ratio of about 1:1.4 protein to sucrose.

[0123] If necessary, the pH of the formulation is set by addition of a
pharmaceutically acceptable acid and/or base. The preferred
pharmaceutically acceptable acid is hydrochloric acid. The preferred base
is sodium hydroxide.

[0124] During formulation development, the stability of the SC drug
product was studied as a function of pH. Example V describes stability
studies with SC Belatacept drug product as a function of pH. The SC
formulation pH was adjusted between 7 to 8.2, samples were placed on
stability and the drug product was monitored for an increase in the high
molecular weight species at various time points using a
stability-indicating size-exclusion chromatography (SE-HPLC) assay. Under
the recommended storage condition of 2°-8° C., no
significant changes in the rate of formation of HMW species were observed
after 3 months.

[0125] In addition to aggregation, deamidation is a common product variant
of peptides and proteins that may occur during fermentation, harvest/cell
clarification, purification, drug substance/drug product storage and
during sample analysis. Deamidation is the loss of NH3 from a
protein forming a succinimide intermediate that can undergo hydrolysis.
The succinimide intermediate results in a 17 u mass decrease of the
parent peptide. The subsequent hydrolysis results in an 18 u mass
increase. Isolation of the succinimide intermediate is difficult due to
instability under aqueous conditions. As such, deamidation is typically
detectable as 1 u mass increase. Deamidation of an asparagine results in
either aspartic or isoaspartic acid. The parameters affecting the rate of
deamidation include pH, temperature, solvent dielectric constant, ionic
strength, primary sequence, local polypeptide conformation and tertiary
structure. The amino acid residues adjacent to Asn in the peptide chain
affect deamidation rates. Gly and Ser following an Asn in protein
sequences results in a higher susceptibility to deamidation.

[0126] Preliminary laboratory scale stability studies suggest that
deamidation will exceed reference levels of the peptide mapping test
method at 24 months using the SC abatacept formulation at pH 7.8. The
data at six months under both 2-8° C. and 25° C. at 60%
humidity showed that the rate of demaidation was lower at pH 7.2 and
higher at pH 8 when compared to the SC abatacept sample at pH 7.8.
Examples IX and XII describe laboratory scale pH studies designed to
evaluate deamidation in SC drug product formulations in the pH range of
6.3 to 7.2.

[0127] The acceptable pH range for the SC drug product is from 6 to 8,
preferably 6 to 7.8, more preferably 6 to 7.2.

[0128] In another aspect, the salts or buffer components may be added in
an amount of at least 10 mM, preferably 10-200 mM. The salts and/or
buffers are pharmaceutically acceptable and are derived from various
known acids (inorganic and organic) with "base forming" metals or amines.
In addition to phosphate buffers, there can be used glycinate, carbonate,
citrate buffers and the like, in which case, sodium, potassium or
ammonium ions can serve as counterion.

[0130] The aqueous carrier of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration to a
human) and is useful for the preparation of a liquid formulation.
Illustrative carriers include sterile water for injection (SWFI),
bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.
phosphate-buffered saline), sterile saline solution, Ringer's solution or
dextrose solution.

[0131] A preservative may be optionally added to the formulations herein
to reduce bacterial action. The addition of a preservative may, for
example, facilitate the production of a multi-use (multiple-dose)
formulation.

[0132] As discussed with the lyophilized drug product, the CTLA4Ig
molecule is incompatible with silicone found in standard syringes, in
that, it interacts with silicone to form visible particulates, thereby
limiting the patient to utilizing silicone free syringes. The SC
formulation may optionally comprise a surfactant to prevent formation of
visible particulates in presence of silicone

[0133] Examples V and VIII describe the effect of surfactants such as
Polysorbate 80 and Poloxamer 188 on the solution stability of belatacept
and abatacept drug product, respectively, and it was found that
surfactants did not, have an effect on the stability of the CTLA4Ig
molecule in a SC formulation. Different levels of Poloxamer 188 were
evaluated and the concentration of from 4 mg/ml to 8 mg/ml, preferable 8
mg/ml was found to be adequate to prevent the silicone related
particulate formation in the formulation.

[0137] The recommended storage condition for the SC formulation is from
2-8° C. with a recommended shelf life of at least 12 months.

[0138] The density of the abatacept SC drug product and the matching
placebo was determined at ambient temperature using the Mettler-Toledo
densitometer. Measurements were performed using 5-mL samples in
triplicates. The density of the abatacept SC formulation was found to be
1.1 g/cc and that of the placebo product was found to be 1.065 g/cc.
Typically, the density of a SC CTLA4Ig formulation is about 1.0 g/cc to
about 1.2 g/cc, preferably about 1.0 g/cc to about 1.15 Wee, more
preferably about 1.095 glee to about 1.105 glee.

[0139] The viscosity of the abatacept SC formulation was determined using
the Brookfield rheometer at ambient temperature. A reference standard of
9.3 cps was used for the measurements. The viscosity of the SC drug
product at 125 mg/mL abatacept concentration was found to be 13±2 cps.
Typically, the viscosity of a SC CTLA4Ig formulation at 125 mg/mL is
about 9 to about 20 cps, preferably about 9 to about 15 cps, more
preferably 12 to about 15 cps.

[0140] Osmolality of SC abatacept drug product and placebo formulation was
measured using a vapor pressure method. The data show that at
concentrations of 125 mg/mL, the osmolality of abatacept SC formulation
is 770±25 mOsm/kgH2O Typically, the osmolatlity of a SC CTLA4Ig
formulation at 125 mg/mL is about 250 to about 800 mOsm/kgH2O,
preferably about 700 to about 800 mOsm/kgH2O, more preferably about
750 to about 800 mOsm/kgH2O.

Preparation of the SC Formulation

[0141] The manufacturing process developed for SC formulations typically
involves compounding with sugar and surfactant, followed by aseptic
sterile filtration and filling into vials or syringes, optionally
preceded by diafiltration (buffer exchange) and concentration of drug
substance using an ultrafiltration unit.

[0142] Examples I and II describe the manufacture of the SC Belatacept and
Abatacept, drug products, respectively.

[0143] One skilled in the art would be aware of the need to overfill the
container so as to compensate for vial, needle, syringe hold-up during
preparation and injection. For example, a 40% overage of drug product is
incorporated into each vial of SC liquid formulation to account for
withdrawal losses and guarantee that 0.8 ml of the solution containing
100 mg of belatacept drug product can be withdrawn from the vial.

Liquid Formulation

[0144] IV formulations may be the preferred administration route in
particular instances, such as when a patient is in the hospital after
transplantation receiving all drugs via the IV route. One skilled in the
art would recognize the disadvantages and risks of a lyophilized
formulation for both the manufacturer and the health care professional,
respectively. The risks and disadvantages to the health care professional
associated with reconstitution can include contamination, foaming and
product loss as well the health care professional's time required to
prepare the IV formulation. Additionally, the manufacturer's costs in
equipment and employee time can be decreased by removing the
lyophilization step of a manufacturing process. All of these reasons are
sufficient motivation to design a liquid formulation for IV use.

[0145] The preferred liquid formulation to develop would be a formulation
that would mimic the lyophilized drug product after the first
constitution to a protein concentration of about 25 mg/ml. The purchased
liquid formulation would then be further diluted to the desired drug
product concentrations between 1 and 10 mg/ml with 0.9% Sodium Chloride
Injection, USP by the health care professional at time of use. The
diluted drug product for injection is isotonic and suitable for
administration by intravenous infusion.

[0146] As discussed above, long term stability of liquid formulations is
an issue for protein drug products. In order to confirm the long term
stability of a solution against formation of high molecular weight
species, formulation development studies were conducted to evaluate the
solution state stability of the liquid formulation of the invention.

[0147] The liquid formulation of the invention comprises the CTLA4Ig
molecule at a protein concentration of at least 20 mg/ml in combination
with a sugar at stabilizing levels, preferably at least 25 mg/ml in
combination with a sugar at stabilizing level in an aqueous carrier.
Preferably the sugar is in a weight ratio of at least 1:1 protein to
sugar. The sugar is preferably disaccharides, most preferably sucrose.
The liquid formulation may also comprise one or more of the components
selected from the list consisting of buffering agents, surfactants, and
preservatives.

[0148] The amount of sucrose useful for stabilization of the liquid drug
product is in a weight ratio of at least 1:1 protein to sucrose,
preferably in a weight ratio of from 1:2 to 1:10 protein to sucrose, more
preferably in a weight ratio of about 1:2 protein to sucrose.

[0149] If necessary, the pH of the formulation is set by addition of a
pharmaceutically acceptable acid and/or base. The preferred
pharmaceutically acceptable acid is hydrochloric acid. The preferred base
is sodium hydroxide.

[0150] During formulation development, the stability of the liquid drug
product was studied at a target pH of 7.5. Example VII describes
stability studies with liquid Belatacept drug product, at a pH of 7.5.
The liquid formulation pH was adjusted to 7.5, samples were placed on
stability and the drug product was monitored for an increase in the high
molecular weight species at various time points using a
stability-indicating size-exclusion chromatography (SE-HPLC) assay. Under
the recommended storage condition of 2°-8° C., no
significant changes in the rate of formation of HMW species were
observed.

[0151] In addition to aggregation, deamidation and fragmentation are
product variant of peptides and proteins that may occur during
fermentation, harvest/cell clarification, purification, drug
substance/drug product storage and during sample analysis. Preliminary
laboratory scale stability studies suggest that deamidation will exceed
reference levels of the peptide mapping test method (rise above the 5%
T26a or T26b (% T30) maximum) and that fragmentation will exceed
reference levels for the SDS-PAGE test method (drop below the 96% major
band minimum) at 24 months using the belatacept (20 mg/ml at pH 7.5)
stored at 2°-8° C. Data from liquid formulations (see SC
data above) show that the rate of deamidation found in the formulations
of the invention decreases as the pH of the formulations is lowered.

[0152] The acceptable pH range for the liquid drug product is from 6 to 8
preferably 6 to 7.8, more preferably 6 to 7.2.

[0153] In another aspect, the salts or buffer components may be added in
an amount of at least about 10 mM, preferably 10-200 mM. The salts and/or
buffers are pharmaceutically acceptable and are derived from various
known acids (inorganic and organic) with "base forming" metals or amines.
In addition to phosphate buffers, there can be used glycinate, carbonate,
citrate buffers and the like, in which case, sodium, potassium or
ammonium ions can serve as counterion.

[0154] The aqueous carrier of interest herein is one which is
pharmaceutically acceptable (safe and non-toxic for administration to a
human) and is useful for the preparation of a liquid formulation.
Illustrative carriers include sterile water for injection (SWFI),
bacteriostatic water for injection (BWFI), a pH buffered solution (e.g.
phosphate-buffered saline), sterile saline solution, Ringer's solution or
dextrose solution.

[0155] A preservative may be optionally added to the formulations herein
to reduce bacterial action. The addition of a preservative may, for
example, facilitate the production of a multi-use (multiple-dose)
formulation.

[0156] As discussed with the lyophilized drug product, the CTLA4Ig
molecule is incompatible with silicone found in standard syringes, in
that, it interacts with silicone to form visible particulates, thereby
limiting the health care professional to utilizing silicone free
syringes. The liquid formulation may optionally comprise a surfactant to
prevent formation of visible particulates in presence of silicone.

[0158] The recommended storage condition for the liquid formulation is
from 2-8° C. with a recommended shelf life at least 12 months.

Preparation of the Liquid Formulation

[0159] The liquid drug product manufacturing process typically involves
compounding with sugar and optionally surfactant followed by aseptic
filtration and filling in vials, stoppering and capping.

[0160] The Formulated Bulk Solution is typically set at a fixed protein
concentration so that the desired vial fill volume can be kept constant.
During the addition of sugar to drug substance, the mixer speed is
controlled at 250±50 rpm so as to minimize foaming in the batching
solution and to ensure complete dissolution of the sugar within 10 to 20
minutes. The Formulated Bulk Solution can be stored under
2°-8° C. or room temperature and room light for at least 24
hours prior to filling.

[0161] The Bulk Solution is not terminally sterilized due to heat
sensitivity of the CTLA4Ig molecule. The Bulk Solution may be sterilized
using two 0.22-μm Millipore Millipak® sterilizing grade filters in
series prior to filling in vials.

[0162] One skilled in the art would be aware of the need to overfill the
container so as to compensate for vial, needle, syringe hold-up during
preparation and injection. For example, each vial of Belatacept drug
product, 20 mg/mL (250 mg/vial), contains a 4% overage of drug product to
account for reconstitution and withdrawal losses.

Articles of Manufacture

[0163] In another embodiment of the invention, an article of manufacture
is provided which contains the drug product and preferably provides
instructions for its use. The article of manufacture comprises a
container. Suitable containers include, for example, bottles, vials,
syringes and test tubes. The container may be formed from a variety of
materials such as glass, plastic or metals.

[0164] The container holds the lyophilized or liquid formulations. The
label on, or associated with, the container may indicate directions for
reconstitution and/or use. For example, the label may indicate that the
25 mg/ml belatacept drug product is to be diluted to protein
concentrations as described above. The label may further indicate that
the SC formulation is useful or intended for subcutaneous administration.
The container holding the formulation may be a multi-use vial, which
allows for repeat administrations (e.g. from 2-6 administrations) of, for
example, the subcutaneous formulation. Alternatively, the container may
be a pre-filled syringe containing, for example, the subcutaneous
formulation.

[0165] The article of manufacture may further comprise a second container
comprising, for example, a suitable carrier for the lyophilized
formulation.

[0166] The article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other buffers,
diluents, filters, needles, syringes, and package inserts with
instructions for use.

[0167] Silicone free syringes are preferably utilized for surfactant free
drug product, such as upon reconstitution of lyophilized drug product
and/or transfer of the solutions from the vial to the intravenous bag and
may be co-packaged with the drug product vial.

[0169] The present invention further provides a method for inhibiting
solid organ and/or tissue transplant rejections by a subject, the subject
being a recepient of transplant tissue. Typically, in tissue transplants,
rejection of the graft is initiated through its recognition as foreign by
T cells, followed by an immune response that destroys the graft. The
CTLA4Ig molecule formulations of this invention, by inhibiting T
lymphocyte proliferation and/or cytokine secretion, may result in reduced
tissue destruction and induction of antigen-specific T cell
unresponsiveness may result in long-term graft acceptance without the
need for generalized immunosuppression. Furthermore, the CTLA4Ig molecule
formulations of the invention can be administered with other
pharmaceuticals including, but not limited to, corticosteroids,
cyclosporine, rapamycin, mycophenolate mofetil, azathioprine,
tacrolismus, basiliximab, and/or other biologics.

[0170] The present invention also provides methods for inhibiting graft
versus host disease in a subject. This method comprises administering to
the subject the formulations of the invention, alone or together, with
further additional ligands, reactive with IL-2, IL-4, or
γ-interferon. For example, a CTLA4Ig molecule SC formulation of
this invention may be administered to a bone marrow transplant recipient
to inhibit the alloreactivity of donor T cells. Alternatively, donor T
cells within a bone marrow graft may be tolerized to a recipient's
alloantigens ex vivo prior to transplantation.

[0171] Inhibition of T cell responses by CTLA4Ig molecule formulations of
the invention may also be useful for treating autoimmune disorders. Many
autoimmune disorders result from inappropriate activation of T cells that
are reactive against autoantigens, and which promote the production of
cytokines and autoantibodies that are involved in the pathology of the
disease. Administration of a CTLA4Ig molecule formulation in a subject
suffering from or susceptible to an autoimmune disorder may prevent the
activation of autoreactive T cells and may reduce or eliminate disease
symptoms. This method may also comprise administering to the subject a
formulation of the invention, alone or together, with further additional
ligands, reactive with IL-2, IL-4, or γ-interferon.

[0172] The most effective mode of administration and dosage regimen for
the formulations of this invention depends upon the severity and course
of the disease, the patient's health and response to treatment and the
judgment of the treating physician. In accordance with the practice of
the invention an effective amount for treating a subject may be between
about 0.1 and about 10 mg/kg body weight of subject. Also, the effective
amount may be an amount between about 1 and about 10 mg/kg body weight of
subject.

[0173] The CTLA4Ig molecule formulations of the invention may be
administered to a subject in an amount and for a time (e.g. length of
time and/or multiple times) sufficient to block endogenous B7 (e.g., CD80
and/or CD86) molecules from binding their respective ligands, in the
subject. Blockage of endogenous B7/ligand binding thereby inhibits
interactions between B7-positive cells (e.g., CD80- and/or CD86-positive
cells) with CD28- and/or CTLA4-positive cells. Dosage of the CTLA4Ig
molecule is dependant upon many factors including, but not limited to,
the type of tissue affected, the type of disease being treated, the
severity of the disease, a subject's health, and a subject's response to
the treatment with the agents. Accordingly, dosages of the agents can
vary depending on the subject and the mode of administration, US patent
application US Publication Number US 2003/0083246 and US patent
application US Publication Number US 2004/0022787 teach dosage and
administration schedules for CTLA4Ig having the amino acid sequence shown
in SEQ ID NO:2 for treating rheumatic diseases, such as rheumatoid
arthritis. All are herein incorporated by reference

[0174] An effective amount of CTLA4Ig molecule may be administered to a
subject daily, weekly, monthly and/or yearly, in single or multiple times
per hour/day/week/month/year, depending on need. For example, in one
embodiment, an effective amount of the CTLA4Ig molecule may initially be
administered once every two weeks for a month, and then once every month
thereafter.

[0175] An effective amount of CTLA4Ig molecule is an amount about 0.1 to
100 mg/kg weight of a subject. In another embodiment, the effective
amount is an amount about 0.1 to 20 mg/kg weight of a subject. In a
specific embodiment, the effective amount of CTLA4Ig is about 2 mg/kg
weight of a subject. In another specific embodiment, the effective amount
of CTLA4Ig is about 10 mg/kg weight of a subject. In another specific
embodiment, an effective amount of CTLA4Ig is 500 mg for a subject
weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg
and 1000 mg for a subject weighing more than 100 kg.

[0177] Typically, doses of the CTLA4Ig molecule formulation of the
invention are based on body weight, and administration regimens may be
dictated by the target serum trough profiles. Typically, target trough
serum concentration of LEA29YIg molecules of the invention between about
3 μg/mL and about 30 μg/mL over the first 3 to 6 months
post-transplant will be sufficient to maintain function of the allograft,
preferably between about 5 μg/mL and about 20 μg/mL. Typically,
target trough serum concentration of LEA29YIg molecules of the invention
during the maintenance phase are between about 0.2 μg/mL and about 3
μg/mL, preferably between about 0.25 μg/mL and about 2.5 μg/mL.

[0178] The LEA29YIg molecules of the invention may be administered in an
amount between about 0.1 to about 20.0 mg/kg weight of the patient,
typically between about 1.0 to about 15.0 mg/kg. For example, L104EA29YIg
may be administered at 10 mg/kg weight of the patient during the early
phase, high risk period that follows transplantation and decreased to 5
mg/kg weight of the patient for a maintenance dosage.

[0179] The administration of the CTLA4Ig molecules of the invention can be
via a 30 minute to one or more hour intravenous infusion. Alternatively,
single to multiple subcutaneous injections can deliver the required
dosage. Typically, a 30 minute intravenous infusion is the administration
route utilized during the early phase of treatment while the patient is
in the hospital and/or making scheduled visits to the healthcare
professional for monitoring. The subcutaneous injection is the typical
administration mode utilized during the maintenance phase, thereby
allowing the patient to return to their normal schedule by decreasing the
visits to a healthcare professional for intravenous infusions.

[0180] Example 10 describes the pharmacokinetics of the lyophilized IV
CTLA4Ig formulation in healthy subjects and patients with rheumatoid
arthritis (RA). The pharmacokinetics of abatacept in RA patients and
healthy subjects appeared to be comparable. In RA patients, after
multiple intravenous infusions, the pharmacokinetics of abatacept showed
proportional increases of Cmax and AUC over the dose range of 2
mg/kg to 10 mg/kg. At 10 mg/kg, serum concentration appeared to reach a
steady-state by day 60 with a mean (range) trough concentration of 24
mcg/mL (from about 1 to about 66 mcg/mL). No systemic accumulation of
abatacept occurred upon continued repeated treatment with 10 mg/kg at
monthly intervals in RA patients.

[0181] The invention will be more fully understood by reference to the
following examples. They should not, however, be construed as limiting
the scope of the invention. All citations throughout the disclosure are
hereby expressly incorporated by reference.

[0184] The manufacturing process for Belatacept SC drug product, 125 mg/ml
(100 mg/vial) drug product involves buffer exchange of the bulk drug
substance from 25 mM sodium phosphate, 10 mM sodium chloride buffer at a
pH of 7.5 to 10 mM sodium phosphate pH 7.5 buffer, followed by
concentration of the protein from ˜25 mg/ml to ˜150 mg/ml by
removal of buffer. The buffer exchange is accomplished by five times
diafiltration of the bulk drug substance against the new 10 mM sodium
phosphate pH 7.5 buffer, followed by concentration of protein to
˜150 mg/ml by removal of buffer. A stainless steel Pelicon mini
filter holder (Millipore) is equipped with stainless steel pressure
gauges and membrane valves on the feed, retentate and permeates port. Two
filtration cassettes used with Pellicon mini module are fitted with 0.1
m2 area Biomax polyethersulfone membrane with 30 kDa nominal
molecular weight cutoff limit. The filtration cassettes are installed
according to the manufacturer's recommendations. The feed container for
the drug substance is a 4 liter glass container with magnetic stir bar.
MasterFlex high performance silicone tubing is used to connect the feed
container to the filter holder and for the permeate line. Feed flow is
provided by peristaltic pump installed in the feed line. Sucrose and
Poloxamer 188 are then dissolved in the concentrated protein solution and
final batch weight is adjusted with 10 mM sodium phosphate buffer, pH
7.5. The bulk solution is filtered through 0.22 micron sterilizing filter
and filled into sterilized and depyrogenated 5-cc Type I flint glass
vials, stoppered with 20 mm rubber stoppers and sealed with 20 mm
aluminum flip-off seals. The composition of Belatacept SC drug product,
125 mg/ml (100 mg/vial) drug product is provided in Table 8 below.

[0188] Abatacept, lyophilized, (250 mg/vial) drug product is a sterile,
non-pyrogenic lyophile suitable for intravenous (IV) administration. Each
single-use vial contains 250 mg of abatacept which is constituted with
Sterile Water for Injection, USP and further diluted with 0.9% Sodium
Chloride Injection, USP, at the time of use.

[0189] The batch formula for a 115 liter batch size is described in Table
11 below.

[0190] The required quantity of abatacept drug substance is added to a
cleaned and sterilized stainless steel compounding vessel equipped with a
mixer. The drug substance solution is mixed at 250±50 rpm while
maintaining the solution temperature between 5°-25° C.

[0191] The required quantity of maltose monohydrate powder is added to the
compounding vessel. The solution is mixed for a minimum of 10 minutes at
15°-25° C.

[0192] The solution pH is adjusted to 7.3-7.7, if necessary using the
previously prepared 1 N sodium hydroxide solution or 1 N hydrochloric
acid solution. The batch is brought to the final batch weight (final
q.s.) using Water for Injection, USP, and mixed for a minimum of 8
minutes. The formulated bulk solution is sampled for pH.

[0193] Formulated Bulk Solution is pre-filtered with one 0.45-μm
filter. The formulated bulk solution after 0.45-μm filter is sampled
for bioburden and bacterial endotoxin (BET).

[0194] The pre-filtered formulated bulk Solution is sterile filtered with
two 0.22-μm filters in series prior to filling.

[0197] At the end of the lyophilization cycle, the chamber pressure is
raised to 500 microns using sterile filtered nitrogen and vial stoppering
is performed under vacuum. The stoppered vials remain inside the
lyophilizer for at least 4 hours. The lyophilized and stoppered vials are
sealed with a 20-mm aluminum, white flip-off seal under HEPA filtered air
by the capping machine. The sealed vials are rinsed with deionized water
by an exterior vial washer. The washed drug product vials are stored at 2
to 8° C.

[0199] Belatacept, lyophilized, (100 mg/vial), drug product is a sterile,
non-pyrogenic lyophile suitable for intravenous (IV) administration. Each
single-use vial contains 100 mg of belatacept with is constituted with
4.2 ml of Sterile Water for Injection, USP to yield a concentration of 25
mg/ml. It can be further diluted to a concentration as low as 1 mg/ml
with 5% Dextrose Injection, USP or 0.9% Sodium Chloride Injection, USP at
the time of use.

[0200] The batch size for drug product manufacture may vary from 20 liters
to 120 liters. A representative batch formula for a batch size of 66
liters (12,000 vials) is provided in Table 14 below.

[0203] Stability studies of the SC liquid formulation of Belatacept drug
product were conducted by placing formulations on stability at different
temperatures and for various time periods.

Effect of Sucrose

[0204] Formulation development studies were conducted to evaluate the
effect of various levels of sucrose on solution stability of belatacept
drug product. Samples were placed on stability at -70° C.,
8° C. and 25° C./60% humidity conditions and monitored at
various time points. The ratios of protein to sucrose evaluated were 1:1,
1:1.7 and 1:1.75. The formation of high molecular weight (HMW) species of
belatacept was utilized to determine protein stability in solution.
Results are shown in Table 16 below.

[0205] The results of the studies showed that increasing the sucrose to
protein ratio improved protein stability. A protein to sucrose ratio of
1:1.36 (wt.:wt.) was chosen for the development of the SC solution
because it provided optimum stability without resulting in drug product
with excessive hypertonicity.

Effect of Surfactants

[0206] The effect of various surfactants in marketed products, such as
Polysorbate 80 and Poloxamer 188 on the solution stability of belatacept
drug product was evaluated. Poloxamer 188 was evaluated at levels of 4, 6
and 8 mg/ml and Polysorbate 80 was evaluated at 1 and 2 mg/ml of final
formulation concentration. Samples were placed on stability at
-70° C., 8° C. and 25° C./60% humidity conditions
and monitored at various time points. Results are shown in Table 17
below.

[0207] Results of the effect of surfactants suggested that surfactant did
not have a significant effect on the stability of belatacept drug product
solution. Among the levels of Poloxamer 188 evaluated. the concentration
of 8 mg/ml was found to be adequate to prevent the formation of silicone
related particulates in the formulation.

Effect of pH

[0208] Stability of the Belatacept SC, (125 mg/ml, protein:sucrose 1:1.36,
8 mg/ml Pluronic F68) drug product was evaluated as a function of pH. The
solution pH was adjusted between 7 to 8.2 with either 1N sodium hydroxide
or 1N Hydrochloric acid. Samples were placed on stability at 2-8°
C. and 25° C./60% RH conditions and monitored at various time
points. Analytical testing included pH and SE-HPLC to monitor increase in
high molecular weight (HMW) species. These results are summarized in
Table 18 below.

[0209] No significant changes in the rate of formation of HMW species were
observed under the recommended storage condition of 2-8° C.
Additionally, the solution-state stability data showed the pH of maximum
stability to be between. 7 and 8. Based on this, a pH range of 7-8 with a
target pH of 7.5 was selected for this formulation.

Osmolality

[0210] Osmolality of belatacept drug product solutions in various buffers,
at different protein concentrations and from separate steps of the
formulation process were measured using a vapor pressure method. These
results are summarized in Table 19 below.

[0211] The effect of agitation on solution stability of belatacept SC drug
product at 100 mg/ml and 125 mg/ml concentration was determined. Aliquots
of the solution containing approximately 1 ml in 5 cc tubing vials were
shaken at speed 3 of wrist arm shaker at 2-8° C. The temperature
of the shaker was maintained at 2-8° C. by placing the shaker in
the cold room. Samples were withdrawn at appropriate time intervals and
assayed for pH and visual appearance, and same time samples were also
evaluated for bioactivity after 30 days of agitation.

[0212] Samples agitated at 100 mg/ml and 125 mg/ml concentration for up to
30 days show no change in the level of HMW species, in SDS-PAGE profile,
peptide mapping, B7 binding assay, pH, appearance or protein
concentration when agitated at 2-8° C.

Effect of Multiple Freeze/Thaw

[0213] The effect of multiple freezing and thawing on stability of
belatacept SC drug product formulation was investigated in samples with
pH ranging from 7.0 to 8.2. Approximately 10 μl aliquots of belatacept
SC drug product formulation (125 mg/ml) at pH 7.0, 7.4, 7.8 and 8.2 were
dispensed into 30 ml Nalgene PETG containers. Multiple freezing and
thawing were performed by storing vials at -70° C. followed by
thawing at ambient temperature (25° C.) for 10 minutes. This cycle
was repeated for 5 days. The contents of vials were analyzed for pH, %
HMW species and appearance after each freeze/thaw cycle.

[0214] No change in pH, appearance or % high molecular weight species
content was observed in samples during five freeze/thaw cycles.

[0216] Syringe-ability study was performed with belatacept SC drug product
(125 mg/ml) at 2°-8° C. condition. Various needle sizes
with 1 ml and 0.5 mL syringe were evaluated. Syringe filling time and
delivery force are recorded in Table 20 below.

[0217] Based on the syringe-ability study results shown in Table 20. A 21
gauge×11/2 inch sterile hypodermic needle is recommended for
withdrawal of this product from vial and a 27 gauge×1/2 inch needle
for subsequent dosing.

Example VI

[0218] Stability studies of the lyophilized formulation of Abatacept drug
product were conducted by placing formulations on stability at different
temperatures and for various time periods.

Effect of Maltose

[0219] Formulation development studies were conducted to evaluate the
effect of various levels of maltose on the stability of abatacept drug
product. Samples were placed on stability at 50° C. and monitored
at various time points. The ratios of protein to maltose evaluated were
1:1, 1:2 and 1:5. The formation of high molecular weight (HMW) species of
abatacept was utilized to determine protein stability in solid state.
Results are shown in Table 21 below.

TABLE-US-00021
TABLE 21
Effect of Maltose on the Freeze-Dried Solid-State
Stability of Abatacept drug product at 50° C.
Level of High Molecular Weight
Species by SE-HPLC (Area %)
Drug to Maltose Weight Ratio
Time (Weeks) Without Maltose 1:1 1:2 1:5a
Initial 0.9 0.7 0.6 2.0
2 5.4 3.7 2.0 NA
4 8.0 5.9 2.7 2.4
6 10.2 6.8 3.3 NA
8 11.7 7.5 3.9 2.9
aStability of drug product with 1:5 drug-to-maltose weight ratio was
evaluated during early development with 50 mg/vial strength. The drug
substance lot used in this study was different from that used for other
results in this table. This is the reason for the different initial
levels of high molecular weight species in these samples.

[0220] The results demonstrate that the stability of abatacept drug
product in a freeze-dried solid-state form is enhanced in the presence of
maltose. Additionally, the minimum amount of maltose useful for
stabilization of abatacept was determined to be at a 1:2 protein to
maltose weight ratio.

Effect of pH

[0221] Stability of lyophilized Abatacept drug product, (250 mg/vial,
protein:maltose 1:2) was evaluated as a function of pH. The solution pH
was adjusted between 6 to 8 with either 1N sodium hydroxide or 1N
Hydrochloric acid. Samples were placed on stability at
2°-8° C. conditions and monitored at various time points.
Analytical testing included pH and SE-HPLC to monitor increase in high
molecular weight (HMW) species. These results are summarized in Table 22
below.

[0222] Under the recommended storage condition of 2°-8° C.,
no significant changes in the rate of formation of HMW species were
observed. Additionally; the solution-state stability data generated
during an early development showed the pH of maximum stability to be
between 7 and 8.

Example VII

[0223] Stability studies of the liquid formulation of belatacept (20
mg/ml) drug product were conducted by placing formulations on stability
at different temperatures and for various time periods.

Effect of Sucrose

[0224] The formulation development studies were conducted to evaluate the
effect of various levels of sucrose on the solution stability of
belatacept liquid drug product at 20 mg/ml. Samples were placed on
stability at 8° C., 25° C./60% humidity and 30°
C./60% humidity conditions and monitored at various time points. The
ratios of protein to sucrose evaluated were 1:1, 1:2, 1:5 and 1:10
protein:sucrose ratio with 20 mg/mL of belatacept. The formation of high
molecular weight (HMW) species of belatacept was utilized to deter mine
protein stability in solution. Results of these study is summarized and
shown in Table 23 below.

[0225] The results of the studies showed that increasing the sucrose to
protein ratio improved protein stability. A protein to sucrose ratio of
1:2 (wt.:wt.) was chosen for the development of the liquid solution
because it provided optimum stability without resulting in drug product
with excessive hypertonicity.

Stability Study

[0226] Three liquid beleatacept batches were prepared and placed on
stability at 2-8° C. and 25° C./60% RH conditions and
monitored at various time points. The weight ratio of protein to sucrose
was 1:2. The formation of high molecular weight (HMW) species of
belatacept was utilized to determine protein stability in liquid
formulation. Stability data is summarized in Table 24 below.

[0227] The data indicate only 0.1% increase in high molecular weight
species in liquid formulation compared to 0.2% increase in Belatacept
drug substance without sucrose in 12 months at 2-8° C. These
results also indicate that addition of sucrose does help in reducing
formation of high molecular weight species.

Example VIII

[0228] Stability studies of the SC formulation of Abatacept drug product
were conducted by placing formulations on stability at different
temperatures and for various time periods.

[0229] Effect of Buffer Strength

[0230] Stability of SC Abatacept drug product, (100 mg/ml) was evaluated
as a function of buffer strength. The buffer system was either 5 or 10 mM
phosphate buffer. Samples were placed on stability at 2-8° C. and
30° C./60% RH conditions and monitored at various time points.
Analytical testing included pH and SE-HPLC to monitor increase in high
molecular weight (HMW) species. These results are summarized in Table 25
below.

[0232] Formulation development studies were conducted to evaluate the
effect of various sugars on solution stability of abatacept SC drug
product. Samples were placed on stability at 2-8° C. and
30° C./60% humidity conditions and monitored at various time
points. The sugars evaluated were sucrose, trehalose and mannitol. The
formation of high molecular weight (HMW) species of abatacept was
utilized to determine protein stability in solution. Results are shown in
Table 26 below.

[0233] The results of the studies showed that all three sugars sucrose,
trehalose and mannitol offered better stabilization to abatacept compared
to the control without sugar. The results of the studies under
accelerated conditions of 30 C showed that mannitol offered better
stabilization to abatacept compared to sucrose and trehalose. Sucrose was
slightly better than trehalose. Under refrigeration, the stabilization by
all three sugards was not significantly different. Sucrose was chosen as
sugar of choice since mannitol formulation had twice the tonicity of the
sucrose formulation. Choosing sucrose for stabilization would allow
addition of twice as much sucrose to achieve the same tonicity as
mannitol at the same ratio but much greater stabilization against
aggregation. A protein:sucrose in ratio of 1:1.36 (wt.:wt.) was chosen
for the development of the SC drug product because it provided optimum
stability without resulting in a drug product with excessive
hypertonicity.

Effect of Sucrose

[0234] Formulation development studies were conducted to evaluate the
effect of various levels of sucrose on solution stability of abatacept SC
drug product. Samples were placed on stability at 2-8° C. and
30° C./60% humidity conditions and monitored at various time
points. The ratios of protein to sucrose evaluated were 1:1 and 1:2. The
formation of high molecular weight (HMW) species of abatacept was
utilized to determine protein stability in solution. Results are shown in
Table 27 below.

[0235] The results of the studies showed that increasing the sucrose to
protein ratio improved protein stability. A protein:sucrose ratio of
1:1.36 (wt.:wt.) was chosen for the development of the RTU solution
because it provided optimum stability without resulting in drug product
with excessive hypertonicity.

Effect of Surfactants

[0236] The effect of various surfactants in marketed products, such as
Polysorbate 80 (Tween® 80) and Poloxamer 188 (Pluronic® F68) on
the solution stability of abatacept SC drag product was evaluated.
Poloxamer 188 was evaluated at levels of 4 and 8 mg/ml and Polysorbate 80
was evaluated at 1 and 2 mg/ml of final formulation concentration.
Samples were placed on stability at -2-8° C. and 25° C./60%
humidity conditions and monitored at various time points. Results are
shown in Table 28 below.

[0237] Results of the effect of surfactants suggested that surfactant did
not have a significant negative effect on the stability of abatacept SC
drug product. Among the levels of Poloxamer 188 evaluated. the
concentration of 8 mg/ml was found to be adequate to prevent the
formation of silicone related particulates in the formulation.

Osmolality

[0238] Osmolality of abatacept in various buffers, at different protein
concentrations and from separate steps of the formulation process were
measured using a vapor pressure method. These results are summarized in
Table 29 below.

[0239] The effect of agitation on solution stability of abatacept SC drag
product at 100 mg/ml and 125 mg/ml concentration was determined. Aliquots
of the solution containing approximately 1 ml in 5 cc tubing vials were
shaken at speed 3 of wrist arm shaker at 2-8° C. The temperature
of the shaker was maintained at 2-8° C. by placing the shaker in
the cold room. Samples were withdrawn at appropriate time intervals and
assayed for pH and visual appearance, and same time samples were also
evaluated for bioactivity after 30 days of agitation.

[0240] Samples agitated at 100 mg/ml and 125 mg/ml concentration for up to
30 days show no change in the level of HMW species, in SDS-PAGE profile,
peptide mapping, B7 binding assay, pH, appearance or protein
concentration when agitated at 2-8° C.

[0242] Deamidation and aggregation are two observed degradation pathways
of CTLA4Ig molecules. This protocol outlines a laboratory scale pH
stability study designed to evaluate the SC drug product formulation in
the pH range of 6.3-7.2, specifically pH 6.3, 6.6, 6.9, 7.2. The purpose
of this study is to identify the optimal lower pH formulation that will
attain a minimum of 18-months of shelf-life for the CTLA4Ig SC
formulations with regards to deamidation and formation of high molecular
weight species. The SC drug product formulation utilized in this study is
described in Table 30 below.

[0243] Abatacept SC drug product will be formulated at pH 6.3, 6.6, 6.9,
7.2. The drug product will be formulated with sucrose and poloxamer 188
as described above and the final batch concentration will be adjusted
with 10 mM phosphate buffer (pH 6.9). The pH will be titrated down to 6.3
and 6.6, respectively, using 1N HCl. Alternatively, the pH will be
titrated up to 6.9, 7.2, and 7.65 with 1N NaOH. The drug product will be
filled into 1-mL long Physiolis® syringes (1.0 ml fill volume) and
placed on stability stations at 2-8° C., 15° C., 25°
C. at 60% humidity, and 35° C. Samples should be protected from
light at all times by covering or inserting into brown light-protective
bags.

[0245] Non-Routine testing methods will be used to further characterize
stability samples at the initial time point, 4 months, 12 months and at
the end of the study. Some of these methods may also be used to test
specific samples if a trend or an unexpected result is observed. The
non-routine methods include: size exclusion chromatography employing
multiangle light scattering (SEC-MALS), kinetic binding (SPR), Mass
Spectrometry, CD, AUC, Differential Scanning calorimeter (DSC), FFF,
FTIR, size exclusion HPLC (denatured) and SDS-PAGE (silver stain).

Example X

[0246] A PK substudy was incorporated in a phase 2B, multi-center,
randomized, double-blind, placebo-controlled study to evaluate the safety
and clinical efficacy of two different doses of abatacept administered
intravenously to subjects with active rheumatoid arthritis while
receiving methotrexate. In this parallel design study, subjects received
abatacept at 2 different doses (2 and 10 mg/kg) or placebo in combination
with MTX. Abatacept was manufactured as described in co-pending U.S.
patent application Ser. No. 60/752,267, filled on Dec. 20, 2005 which
teaches processes for the production of proteins of the invention,
specifically recombinant glycoprotein products, by animal or mammalian
cell cultures, and supplied in lyophilized form, as described herein, in
individual vials containing 200 mg of abatacept. Abatacept was
administered IV to subjects on Days 1, 15, and 30, and every 30 days
thereafter for a year. Multiple dose PK was derived from the serum
concentration vs time data obtained during the dosing interval between
Days 60 and 90 from subjects who were enrolled into a site-specific PK
substudy. For the subjects in the PK substudy, blood samples were
collected before dosing on Day 60, and for a PK profile beginning on Day
60 at 30 minutes (corresponding to the end of abatacept infusion), at 4
hours after the start of infusion, and weekly thereafter until Day 90. A
total of 90 subjects were enrolled to participate in the PK substudy.
However, complete PK profiles between the dosing interval from Day 60 to
90 were obtained from 29 subjects (15 subjects dosed at 2 mg/kg; 14
subjects dosed at 10 mg/kg).

[0247] A summary of the PK parameters is presented in Table 31. The
results from the study showed that both Cmax and AUC(TAU), where TAU=30
days, increased in a dose proportional manner. For nominal doses
increasing in the ratio of 1:5, the geometric means of Cmax increased in
the ratio of 1:5.2, while the geometric mean for AUC(TAU) increased in
the ratio of 1:5.0. In addition, T-HALF, CLT, and Vss values appeared to
be independent of dose. In these RA subjects, the mean T-HALF, CLT, and
Vss values were around 13 days, ˜0.2 mL/h/kg, and ˜0.07 L/kg,
respectively. The small Vss indicates that abatacept is confined
primarily to the extracellular fluid volume. Based on the dosing schema
of dosing at 2 and 4 weeks after the first infusion, then once a month
thereafter, steady-state conditions for abatacept were reached by the
third monthly dose. Also, as a result of the dosing schema, serum
concentrations were above steady-state trough concentrations during the
first 2 months of treatment. Comparison of the trough (Cmin) values at
Days 60, 90, and 180 indicated that abatacept does not appear to
accumulate following monthly dosing. The mean Cmin steady-state values
for all subjects receiving monthly TV doses of 2 and 10 mg/kg abatacept
ranged between 4.4 to 6.7 mcg/mL and 22.0 to 28.7 mcg/mL, respectively.

[0248] The pharmacokinetics of abatacept were studied in healthy adult
subjects after a single 10 mg/kg intravenous infusion and in RA patients
after multiple 10 mg/kg intravenous infusions (see Table 32).

[0249] The pharmacokinetics of abatacept in RA patients and healthy
subjects appeared to be comparable. In RA patients, after multiple
intravenous infusions, the pharmacokinetics of abatacept showed
proportional increases of C and AUC over the dose range of 2 mg/kg to 10
mg/kg. At 10 mg/kg, serum concentration appeared to reach a steady-state
by day 60 with a mean (range) trough concentration of 24 (1-66) mcg/mL.
No systemic accumulation of abatacept occurred upon continued repeated
treatment with 10 mg/kg at monthly intervals in RA patients.

[0250] Population pharmacokinetic analyses in RA patients revealed that
there was a trend toward higher clearance of abatacept with increasing
body weight. Age and gender (when corrected for body weight) did not
affect clearance. Concomitant methotrexate (MTX), nonsteroidal
anti-inflammatory drugs (NSAIDs), corticosteroids, and TNF blocking
agents did not influence abatacept clearance.

Serum Assay for Abatacept

[0251] Serum samples were analyzed for abatacept by an enzyme-linked
immunosorbent assay (ELISA) in a total of 25 analytical runs. All
analytical results met the acceptance criteria established prior to
sample analysis indicating that the ELISA method was precise and accurate
for the quantitation of abatacept in study samples. A summary of the
standard curve parameters and mean QC data for abatacept in serum are
presented in Table 33. The between- and within-run variability of the
analytical QCs for abatacept was 4.5% and 3.5% CV, respectively. Mean
observed concentrations of the analytical QC samples deviated less than
±8.9% from the nominal values (Table 33).

[0252] The objectives of this study are to assess the PK of belatacept
following a single SC dose in the range of 50 to 150 mg in healthy
subjects; to assess the effects of the injection volume and concentration
of the injected solution on the PK of subcutaneously administered
belatacept; to assess the safety and tolerability (including the site of
injection evaluation) of a single SC dose of belatacept; to assess the
immunogenicity of subcutaneously administered belatacept.

[0253] This is a double-blind, randomized, placebo-controlled, parallel
group, single-dose study in healthy subjects. A total of 42 subjects will
be randomized to one of 6 treatment groups. Within each group of 7
subjects, subjects will be randomized in a 5:2 ratio on Day 1 to receive
a single, SC injection of belatacept or placebo. Subjects will be
required to weigh ≦100 kg. The 6 treatment groups are described in
Table 34.

[0254] Subjects will undergo screening evaluations to determine
eligibility within 28 days of dosing on Day 1. Subjects will be admitted
to the clinical facility the day prior to dosing (Day -1) for baseline
evaluations, including MLR. Subjects will remain in the clinical facility
until completion of post-treatment assessments on Day 5, and will return
to the clinical facility for each study visit thereafter until discharged
from the study.

[0255] On Day 1 subjects will be randomized to treatment and will receive
a single SC dose of belatacept or placebo, and undergo detailed PK and
immunogenicity sampling. All subjects will receive the SC injections in
their anterior thigh. Following study drug administration, the
Investigator will assess the injection site for signs of local irritation
and inflammation.

[0256] Physical examinations, vital sign measurements, and clinical
laboratory evaluations will be performed at selected times throughout the
study. Blood samples will be collected for up to 56 days after study drug
administration for PK analysis and assessment of immunogenicity. Subjects
will be monitored for AEs throughout the study. Approximately 265 mL of
blood will be drawn from each subject during the study.

[0257] Dosing and follow-up will occur concurrently for all dose groups.
No subject will receive more than a single dose. Subjects who do not
complete the study (except those who are discontinued for AEs) may be
replaced.

[0258] This is a single dose study. Each subject will undergo a screening
period which will be a maximum of 28 days prior to the day study drug is
administered. Each subject will remain in the study until the last visit,
56 Days (±2 Days) after study drug is administered. The last visit of
the last subject undergoing the trial will be considered the end of the
study.

[0259] Belatacept 100 mg/vial (125 mg/mL), as described herein, and
manufactured as described in co-pending U.S. patent application Ser. No.
60/849,543, filled on Oct. 5, 2006 which teaches processes for the
production of proteins of the invention, specifically recombinant
glycoprotein products, by animal or mammalian cell cultures, is a
ready-to-use liquid product provided in a glass vial for withdrawal and
administration using a suitable size conventional syringe and needle for
SC administration. A sufficient excess of belatacept is incorporated into
each vial to account for withdrawal losses so that 0.8 mL of the solution
containing 100 mg can be withdrawn for SC administration.

[0261] Healthy subjects as determined by medical history, physical
examination, 12-lead electrocardiogram, and clinical laboratory
evaluations will be eligible to participate in the study. This study will
include men and women. Subjects must be at least 18 years of age and
weigh ≦100 kg at the time of randomization. Female subjects must
be not nursing, not pregnant and must be using an acceptable method of
contraception for at least 1 month before dosing, during the study and
for up to 4 weeks after the end of the study. Women of childbearing
potential must have a negative serum pregnancy test within 24 hours prior
to the dose of study medication. Subjects will be advised on potential
risks to a pregnancy. Male subjects must be using an adequate method of
contraception during the study and for up to 4 weeks after the end of the
study so that the risk of pregnancy to their partner is minimized. See
Section 5 for a detailed list of the inclusion and exclusion criteria.

[0262] Medications taken within 4 weeks prior to enrollment must be
recorded on the CRF. No concomitant medications (prescription,
over-the-counter or herbal) are to be administered during study, except
for oral contraceptives, unless they are prescribed by the Investigator
for treatment of specific clinical events. Any concomitant therapies must
be recorded on the CRF.

[0263] PK of belatacept following SC injection will be derived from serum
concentration versus time data. The single-dose PK parameters to be
assessed include:

[0264] Cmax Maximum observed serum concentration

[0265]
Tmax Time of maximum observed serum concentration

[0266] AUC(0-T) Area
under the serum concentration-time curve from time zero to the time of
the last quantifiable concentration

[0267] AUC(INF) Area under the serum
concentration-time curve from time zero extrapolated to infinite time

[0272] Serum samples will be collected over time and assayed for the
presence of antibody titers to belatacept using two ELISA assays. One
assay assesses the response to the whole molecule and the other to the
LEA29Y-T portion only.

[0273] All subjects who receive study medication will be included in the
safety and PD data sets. Subjects who receive placebo in any panel will
be pooled into a single placebo treatment group for PD assessments and
safety assessments except for the site of injection assessments. All
available data from subjects who receive belatacept will be included in
the PK data set, and will be included in the summary statistics and
statistical analysis.

[0274] Baseline is considered Day -1. Frequency distributions of gender
and race will be tabulated by treatment (injection volume and dose).
Summary statistics for age, body weight, and height will be tabulated by
treatment.

[0275] All recorded AEs will be listed and tabulated by preferred term,
system organ class, and treatment. Vital signs and clinical laboratory
test results will be listed and summarized by treatment. Any significant
physical examination findings and clinical laboratory results will be
listed. Injection site assessments (erythema, heat, swelling, pain and
pruritus) will be tabulated by treatment and degree of severity. Placebo
subjects will be pooled across dose groups and analyzed independently, as
well, for the assessment of site of injection.

[0276] Summary statistics will be tabulated for the PK parameters by
treatment. Geometric means and coefficients of variation will be
presented for Cmax, AUC(O-T), and AUC(INF). Medians, minima, and maxima
will be presented for Tmax. Means and standard deviations will be
provided for other PK parameters. To assess the dependency on dose after
SC administration, scatter plots of Cmax and AUC(INF) versus dose will be
provided. Scatter plots of AUC(INF) and Cmax across injection volumes,
will be constructed to assess this effect on the PK of belatacept. Also,
scatter plots of Cmax and AUC(INF) versus dose for a fixed volume, and
Cmax and AUC(INF) versus volume within doses will be provided where
applicable.

[0277] Summary statistics will be tabulated by treatment and study day for
anti-belatacept and anti-LEA29Y-T antibody values and their changes from
baseline (Day 1--0 hr). To explore possible associations between
immunogenicity and exposure, plots of changes in anti-belatacept and
anti-LEA29Y-T antibodies versus belatacept concentrations will be
provided.

[0279] Table 35 above lists the sampling schedule to be followed for the
assessment of PK. Blood samples (˜3 nit per sample) will be
collected into a pre-labeled, red and gray top (SST) Vacutainer tube via
direct venipuncture or from a saline lock. If a saline lock system is
used for blood collection, approximately 0.5 mL of blood should be
withdrawn through the indwelling catheter and be discarded prior to
obtaining each PK sample. Once the PK specimen has been obtained, the
blood will be allowed to clot in the Vacutainer® tube at room
temperature for 15-30 minutes. Following clotting, the sample must be
centrifuged for 15 minutes at 1500×g in a refrigerated centrifuge
(4° C.). When centrifugation is complete, at least 0.5 mL of serum
from each PK sample time point should be removed by pipette and
transferred to a prelabeled, screw cap, polypropylene, PK storage and
shipping tube. A clean pipette must be used to aliquot serum for each
sample time point. The polypropylene tube containing the PK serum sample
may be stored frozen at -20° C. or colder for a maximum of one
month and then at -70° C., thereafter. The time permitted from
sample collection to freezing of the serum is 12 hours. A sensitive,
validated enzyme immunoassay (ETA) method will be used to measure
concentrations of belatacept in serum.

[0280] Table 35 lists the sampling schedule to be followed for the
assessment of immunogenicity. Serum samples will be obtained at visits
Days 1, 14, 28, 35, 42, and 56. The Day 1 immunogenicity
(anti-belatacept) sample should be taken prior to administering study
drug. Samples will be assayed for the presence of anti-belatacept and
anti-LEA29Y-T antibodies. For each specimen, blood (˜3 mL per
sample) will be collected into a pre-labeled red and gray top (SST)
Vacutainer® tube via direct venipuncture or from a saline lock
(indwelling catheter). If a saline lock system is used for blood
collection, approximately 0.5 mL of blood should be drawn through the
indwelling catheter and be discarded prior to obtaining each
immunogenicity sample. Once the specimen has been obtained, the blood
will be allowed to clot in the Vacutainer® tube at room temperature
for 15-30 minutes. Following clotting, the sample must be centrifuged for
15 minutes at 1500×g in a refrigerated centrifuge (4° C.)
When centrifugation is complete, at least 1 mL of serum should be removed
by pipette and transferred to a prelabeled, screw cap, polypropylene,
serum sample storage and shipping tube. The polypropylene tube containing
the serum sample must be stored frozen at -20° C. or colder. Two
sensitive, validated enzyme linked immunosorbent assay (ELISA) methods
will be used to measure antibody titers to belatacept in serum. One assay
assess the antibody titer to the whole molecule and the other to the
LEA29Y-T portion only.

Example XII

[0281] Deamidation, fragmentation and aggregation are observed degradation
pathways of LEA29YIg molecules. This protocol outlines an additional
laboratory scale pH stability study designed to evaluate the belatacept
SC drug product formulation in the pH range of 6.3-7.5. The purpose of
this study is to identify the optimal pH formulation that will attain a
minimum of 18-months of shelf-life for the belatacept SC formulation.

[0282] For this study, belatacept SC product will be formulated at pH 6.3,
6.6, 6.9, 7.2 and 7.5. The belatacept drug substance at ˜25 mg/mL
will be first concentrated to ˜100 mg/mL, then diafiltered into 10
mM phosphate buffer at pH 6.9 followed by second concentration to obtain
a drug product intermediate (DPI) at >160 mg/mL. The DPI will be
formulated with sucrose and poloxamer 188 and the final batch
concentration will be adjusted with 10 mM phosphate buffer (pH 6.9). The
formulated bulk will be subdivided into five sub-batches, as outlined in
the study design. The pH of the sub-batches will be titrated down to 6.3
and 6.6, respectively, using 1N HCl. The pH of the additional two
sub-batches will be titrated up to 6.9, 7.2 and 7.5 with 1N NaOH. The
product batches will be filled into 1-mL long Physiolis®. Samples
should be protected from light at all times by covering or inserting into
brown light-protective bags. The SC drug product formulation utilized in
this study is described in Table 36 below.

[0284] Non-Routine testing methods will be used to further characterize
stability samples at the initial time point, 4 months, 12 months and at
the end of the study. Some of these methods may also be used to test
specific samples if a trend or an unexpected result is observed. The
non-routine methods include: size exclusion chromatography employing
multiangle light scattering (SEC-MALS), kinetic binding (SPR), Mass
Spectrometry, CD, AUC, Differential Scanning calorimeter (DSC), FFF,
FTIR, size exclusion-HPLC (denatured) and SDS-PAGE (silver stain).